**indigenous rhizobia**

microorganisms in the soil. For that reason, hairy vetch cannot easily utilize soil nitrogen under low temperature conditions. On the other hand, it is considered that nitrogen deficiency is related to the poor nodule formation and low nitrogen fixation activity of the rhizobia in the nodules. It is assumed that there is no rhizobia compatible with hairy vetch in the soil or some indigenous rhizobia show low nitrogen fixing activity under low temperature conditions in

*Rhizobium* is a genus of Gram-negative soil bacteria and universally survives in a soil. Rhi‐ zobium colonizes in the leguminous plant cells within root nodules, and fixes atmospheric dinitrogen (N2) to ammonia. Rhizobium provides ammonia as nitrogen source to the host plant, and the host plant provides the rhizobium (bacteroid) organic compounds made by photosynthesis. There is strong host specificity in the symbiosis between rhizobia and legumes. Hairy vetch establishes symbiosis with *Rhizobium leguminosarum* bv. *viciae* and conducts N2 fixation by the nodules. The physiological and genetic diversity of rhizobium is very compli‐ cated in the soil [14]. If hairy vetch is infected by a rhizobium that has low affinity for hairy vetch or has low N2 fixation activity, the host plant hairy vetch shows very poor plant growth. So, for good hairy vetch growth it is important to isolate a superior hairy vetch rhizobium and

Several hairy vetch rhizobium had been isolated from a heavy soil of the upland field converted from a paddy field in Hachirougata polder, Akita Japan. A superior hairy vetch rhizobium was obtained from the isolates and named Y629. The superior hairy vetch rhizobium Y629 was

Seeds of hairy vetch were sown in vermiculite medium supplied with nitrogen free culture solution with inoculation of Y629 or indigenous rhizobia. The hairy vetch plants were grown in a growth chamber with high temperature treatment (day:22°C-12 h, night:18°C -12h) or low

Hairy vetch plants with inoculation of Y629 normally grew, whereas the plants with inocula‐ tion of indigenous rhizobia appeared nutritional disorder irrespective of temperature with poor plant growth (Fig. 3). In high temperature treatment, the nodule number with inoculation of Y629 was fewer than that with inoculation of indigenous rhizobia. However, the nitrogen fixation activity (ARA) per plant with inoculation of Y629 was about two times higher than that with inoculation of indigenous rhizobia. The nitrogen fixation activity with inoculation of Y629 was high even in low temperature condition. These results indicate that the superior hairy vetch rhizobium Y629 shows high nitrogen fixation activity, and Y629 is considered to

identified *Rhizobium leguminosarum* bv. *viciae* by 16S-rRNA gene analysis.

**4. Effect of inoculation of Y629 on hairy vetch growth**

northeast area of Japan.

228 Advances in Biology and Ecology of Nitrogen Fixation

**3. Isolation of a superior hairy vetch rhizobium**

inoculate to the hairy vetch with a superior rhizobium.

temperature treatment (day;15°C -12 h, night;7°C -12h).

have low temperature tolerance in a symbiotic state.

### **Inoculated with Inoculated with Y629**

**Figure 3.** Hairy vetch inoculated with Y629 or indigenous rhizobia at high temperature treatment (Day:22ºC 12h, night: 18ºC 12h).

The nodulation was observed in all the plants, but some nodules with inoculation of indige‐ nous rhizobia showed dark greenish color inside. It has been known that leghemoglobin, a red hemeprotein specifically accumulated in nodules, is an important role in nitrogen fixation in which protects nitrogenase from inactivation by molecular oxygen (O2) [15]. The concentration of leghemoglobin in the nodule is an indicator of nitrogen fixation activity. The section of all the nodules inoculated with Y629 showed red color, which is considered as the accumulation of normal leghemoglobin in the infected region of the nodules. However, some nodules inoculated with indigenous rhizobia exhibited dark color, and those sections showing dark greenish color in the infected region of the nodules which is derived from the decomposition product of leghemoglobin (Fig. 4). The nodules showed dark color might have low or no nitrogen fixation activity. This phenomenon is considered to be an early senescence of nodules caused by the end of symbiosis. It is assumed that rhizobium harbored in the dark color nodules have low affinity for the host plant. It is interesting to note that some rhizobium can infect and form a nodule with host plant, but cannot maintain symbiosis with their host plants.

**Figure 4 Nodulation and section of nodule of hairy vetch inoculated with Y629 or indigenous rhizobia at high temperature treatment (Day:22ºC 12h, Figure 4.** Nodulation and section of nodule of hairy vetch inoculated with Y629 or indigenous rhizobia at high tem‐ perature treatment (Day: 22°C 12h, night: 18°C 12h)

#### **5. Effect of inoculation of Y629 in a field**

**night: 18ºC 12h).**

An upland field converted from a paddy field in Hachirougata polder, Japan was used for this field experiment. The soil type is heavy clay soil and pH is 6.5. There is no hairy vetch planting history in the field. Seeds of hairy vetch were sown with or without inoculation of Y629 in early autumn. Rhizobium Y629 was cultured in YM broth at 30 ºC for 3 days. The culture solution was mixed with vermiculite and peat-moss, and then the seeds of hairy vetch were coated by Y629 mixture. The sowing density was 30 kg ha-1 that is about 200 seeds m-2.

In the field experiment, there was no outstanding effect of Y629 inoculation on the hairy vetch growth before winter. In early spring, the stem length with inoculation of Y629 was as long as that without inoculation. However, about 10 % of the hairy vetch showed nutritional disorder in the non-inoculation treatment. It is supposed that the hairy vetch inoculated with Y629 was avoided nitrogen deficiency according to high nitrogen fixation activity by the nodules infected with Y629. In non-inoculation treatment, some hairy vetch plants were infected with indige‐ nous rhizobia, which might have low nitrogen fixation activity or low affinity for the host plant.

caused by the end of symbiosis. It is assumed that rhizobium harbored in the dark color nodules have low affinity for the host plant. It is interesting to note that some rhizobium can infect and form a nodule with host plant, but cannot maintain symbiosis with their host plants.

**Inoculated indigenous Rhizobia Inoculated Y629**

**night: 18ºC 12h).**

perature treatment (Day: 22°C 12h, night: 18°C 12h)

230 Advances in Biology and Ecology of Nitrogen Fixation

**5. Effect of inoculation of Y629 in a field**

**Figure 4 Nodulation and section of nodule of hairy vetch inoculated with Y629 or indigenous rhizobia at high temperature treatment (Day:22ºC 12h,** 

**Figure 4.** Nodulation and section of nodule of hairy vetch inoculated with Y629 or indigenous rhizobia at high tem‐

An upland field converted from a paddy field in Hachirougata polder, Japan was used for this field experiment. The soil type is heavy clay soil and pH is 6.5. There is no hairy vetch planting history in the field. Seeds of hairy vetch were sown with or without inoculation of Y629 in early autumn. Rhizobium Y629 was cultured in YM broth at 30 ºC for 3 days. The culture solution was mixed with vermiculite and peat-moss, and then the seeds of hairy vetch were coated by Y629 mixture. The sowing density was 30 kg ha-1 that is about 200 seeds m-2.

In the field experiment, there was no outstanding effect of Y629 inoculation on the hairy vetch growth before winter. In early spring, the stem length with inoculation of Y629 was as long as that without inoculation. However, about 10 % of the hairy vetch showed nutritional disorder

The stem length of the plants inoculated with Y629 was slightly higher than those without inoculation in early summer. The dry weight of the shoots with inoculation of Y629 was significantly high compared with non-inoculation treatment. It is confirmed that the hairy vetch growth is promoted by high nitrogen fixation activity by the nodules infected with Y629 under field conditions.

There are many kinds of rhizobia which can establish symbiosis with hairy vetch in the soil of the experimental field. The infection ratio of Y629 with the inoculation method in this experi‐ ment might be below 10 % under the field condition where indigenous hairy vetch rhizobia exist in the soil. It is important that the effect of Y629 inoculation on hairy vetch growth is observed in the field condition even if indigenous rhizobia compatible with hairy vetch exist in the soil (Fig. 5).

**Figure 5 Hairy vetch inoculated with or without Y629 under field condition at Figure 5.** Hairy vetch inoculated with or without Y629 under field condition at 180 days after sowing (15 May 2007).

#### **6. Rhizobium inoculation using micro zeolite powder**

**180 days after sowing (15 May 2007).**

It has been considered that infection ratio of inoculant rhizobium to host leguminous plant is very low in a field condition. In general, commercial rhizobium inoculant product is peat mossbased things, and leguminous crop seeds are usually inoculated mixing with rhizobium inoculant prior to sowing. In this study, the infection ratio of Y629 with the seed inoculation might be below 10 % under the field condition where indigenous hairy vetch rhizobia exist in the soil as described above.

On the other hand, it is necessary to inoculate rhizobium just before sowing, because the population of the inoculated rhizobium on the seed decreased immediately due to drought stress [16]. Thus, it is important for increase in infection ratio of inoculated rhizobium to have high rhizobium population on the seed and to maintain high population of rhizobium until sowing.

The micro zeolite powder of about 0.003 mm in diameter was used for the carrier material of rhizobium inoculant instead of peat moss in the next study. The Y629 YM culture solution at a density of about 108 cells mL-1 was mixed with the micro zeolite powder at a rate of 1:1 (volume ratio). Then, 100 mL of the rhizobium inoculant was mixed with 5 kg of a hairy vetch seeds, and the inoculated seeds were air dried for 1 hour (Fig. 6). The inoculated seeds were

**Figure 6.** Photo of hairy vetch seeds and seeds inoculated with rhizobium (Y629) using the micro zeolite powder.

sown to a field with density of 30 kg ha-1. On the other hand, the inoculated seeds were stored at room temperature with dry condition for 3 month, and then sown to a vermiculite medium filled with nitrogen free culture solution.

inoculant prior to sowing. In this study, the infection ratio of Y629 with the seed inoculation might be below 10 % under the field condition where indigenous hairy vetch rhizobia exist in

On the other hand, it is necessary to inoculate rhizobium just before sowing, because the population of the inoculated rhizobium on the seed decreased immediately due to drought stress [16]. Thus, it is important for increase in infection ratio of inoculated rhizobium to have high rhizobium population on the seed and to maintain high population of rhizobium until

The micro zeolite powder of about 0.003 mm in diameter was used for the carrier material of rhizobium inoculant instead of peat moss in the next study. The Y629 YM culture solution at

(volume ratio). Then, 100 mL of the rhizobium inoculant was mixed with 5 kg of a hairy vetch seeds, and the inoculated seeds were air dried for 1 hour (Fig. 6). The inoculated seeds were

**Figure 6.** Photo of hairy vetch seeds and seeds inoculated with rhizobium (Y629) using the micro zeolite powder.

cells mL-1 was mixed with the micro zeolite powder at a rate of 1:1

the soil as described above.

232 Advances in Biology and Ecology of Nitrogen Fixation

a density of about 108

sowing.

The hairy vetch plant was collected from the field at 30 days after sowing, and the rhizobium was isolated from the nodules. The inoculation rhizobium (Y629) was identified by a genotype. The infection ratio of Y629 was about 30 % under the field condition, whereas that with peat moss-based inoculation might be below 10 % under same condition. The rhizobium population on the seeds with the micro zeolite powder inoculant was as high as with peat moss-based inoculant just after the inoculation. The hairy vetch seed takes about ten days for germination under field condition. In the field experiment in this study, the hairy vetch seeds were not conducted soil cover. Therefore, the rhizobium on the seed surface was got drought stress until germination. The rhizobium with the micro zeolite powder might alive on the seed surface in spite of under drought stress, although the reason has not clear yet. The pH of Y629 culture solution and the micro zeolite powder are about 3 and 10, respectively. It was supposed that the inoculant was neutralized by mixing the rhizobium culture solution and the micro zeolite powder, and the rhizobium was able to maintain population due to improvement of condition to alive on the seed surface. Furthermore, the rhizobium with the micro zeolite powder maintained population enough to form the nodules for 3 month after the inoculation under room temperature with dry condition. The rhizobium inoculant with the micro zeolite powder may be considered to make the rhizobium drought stress tolerant.

#### **7. Flow inoculation of rhizobium in paddy and upland rotation system**

Hairy vetch has been used for nutrient management as green manure to increase in soil fertility and to amend soil physical properties in paddy field and upland field. The rhizobial seed inoculation is troublesome when the cultivation area is very large and the seed amount is a lot. Furthermore, the host plants may not formed nodules even if the seeds are inoculated with rhizobium, when the method of the inoculation was inappropriate or the climatic conditions are not appropriate for rhizobium survival in the soil. Thus, it is necessary to develop new and easy method for rhizobium inoculation in paddy and upland rotation system.

In a paddy field (1.25 ha), 20 L of the Y629 YM broth culture solution at a density of about 108 cells mL-1 was applied with the irrigation water in summer (Fig. 7). The irrigation water was supplied to the field with flow quantity of about 100 m3 per hour. The application speed of the rhizobium solution was about 5 L per hour. After the flow inoculation, conventional farm managementwas carriedoutuntilhairyvetchsowing.Thehairyvetchseedsweresownwithout seed inoculation to the field with density of 30 kg ha-1 at 50 days after the flow inoculation.

The hairy vetch with the flow inoculation formed nodules as well as seed inoculation. The inoculant rhizobium (Y629) population in the soil increased drastically compared with that without flow inoculation treatment. It is considered that the inoculant rhizobium (Y629) could propagate in the soil of the paddy field. The infection ratio of Y629 was about 50 % even if indigenous rhizobia compatible with hairy vetch exist in the soil. In addition, the hairy vetch

**Figure 7.** Photos of inoculant setting on the water intake of the paddy field (Left) and applying of rhizobium (Y629) culture solution (Right) with the flow inoculation.

plant growth with the flow inoculation was promoted compared with the seed inoculation treatment. It is supposed that the rhizobium infection to the hairy vetch root with the flow inoculation was faster than that with seed inoculation because the inoculant rhizobium population was very high in the soil surface. Consequently, the flow inoculation of rhizobi‐ um was considered to be effective to improve the infection ratio of inoculant rhizobium by simple treatment.

#### **8. Conclusion**

Hairy vetch is useful for improving soil structure due to their deep root system, increasing soil fertility and weeding for subsequent crops as described above. Hairy vetch is planted in late summer or autumn, and grows until late autumn. The seedling can survive under the snow in winter and then grow vigorously from spring to early summer. If there is no compatible rhizobium strains suitable for hairy vetch in a soil, inoculation with superior rhizobium such as Y629 bring a significant result on promoting hairy vetch growth. The seed inoculation of rhizobium Y629 using the micro zeolite powder is able to keep rhizobium population on the seed surface after the inoculation possibly by protecting from drought stress. The flow inoculation of rhizobium Y629 is simple and effective method to improve the infection ratio of inoculant rhizobium and to promote the hairy vetch growth under field condition.

#### **Author details**

Takashi Sato

Faculty of Bioresource Sciences Akita Prefectural University, Kaidobata-Nishi, Shimoshinjo-Nakano, Akita, Japan

#### **References**

plant growth with the flow inoculation was promoted compared with the seed inoculation treatment. It is supposed that the rhizobium infection to the hairy vetch root with the flow inoculation was faster than that with seed inoculation because the inoculant rhizobium population was very high in the soil surface. Consequently, the flow inoculation of rhizobi‐ um was considered to be effective to improve the infection ratio of inoculant rhizobium by

**Figure 7.** Photos of inoculant setting on the water intake of the paddy field (Left) and applying of rhizobium (Y629)

Hairy vetch is useful for improving soil structure due to their deep root system, increasing soil fertility and weeding for subsequent crops as described above. Hairy vetch is planted in late summer or autumn, and grows until late autumn. The seedling can survive under the snow in winter and then grow vigorously from spring to early summer. If there is no compatible rhizobium strains suitable for hairy vetch in a soil, inoculation with superior rhizobium such as Y629 bring a significant result on promoting hairy vetch growth. The seed inoculation of rhizobium Y629 using the micro zeolite powder is able to keep rhizobium population on the seed surface after the inoculation possibly by protecting from drought stress. The flow inoculation of rhizobium Y629 is simple and effective method to improve the infection ratio

of inoculant rhizobium and to promote the hairy vetch growth under field condition.

Faculty of Bioresource Sciences Akita Prefectural University, Kaidobata-Nishi, Shimoshinjo-

simple treatment.

culture solution (Right) with the flow inoculation.

234 Advances in Biology and Ecology of Nitrogen Fixation

**8. Conclusion**

**Author details**

Nakano, Akita, Japan

Takashi Sato


## **Role of Boron Nutrient in Nodules Growth and Nitrogen Fixation in Soybean Genotypes Under Water Stress Conditions**

Nacer Bellaloui, Alemu Mengistu, My Abdelmajid Kassem, Craig A. Abel and L.H.S. Zobiole

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56994

**1. Introduction**

[14] Palmer K. M., J. Young, P. W. Higher Diversity of *Rhizobium leguminosarum* biovar *viciae* populations in arable soils than in grass soils, Appl. Environ. Microbiol., 2000;

[15] Appleby C.A. Leghemoglobin and Rhizobium Respiration. Annu. Rev. Plant Physi‐

[16] Rosalind D., Rodney J. R., Ivan R. K. Legume seed inoculation technology—a review.

ol., 1984; 35, 443-478. Varia. Soil Sci. Plant Nutr. 55, 235-242.

Soil Biology and Biochemistry, 2004; 36, 1275–1288.

66, 2445–2450

236 Advances in Biology and Ecology of Nitrogen Fixation

Boron is an essential nutrient for plant growth, development, and seed quality [1-4]. Previous research indicated the involvement of B in cell wall structure [5,6]; cell membrane integrity [2, 7]; sugar metabolism [2], especially sugar alcohols [3,8]; nitrogen assimilation and fixation [9, 10]; nodules [11,12], nodullin protein (ENOD2) and malfunction of oxygen diffusion barrier [13]; phenolic metabolism [2,14,15]; ion uptake [2,16]; and plasma membrane-bount H+ ATPase [7,17,18].

Boron is required for nodules growth and nitrogen fixation [9,11-13], and boron deficiency can occur under certain environmental stress factors even when boron level in soil is adequate [19], leading to yield loss. Among the environmental stress factors that can lead to boron deficiency in plants is drought or water stress. Drought is a major environmental stress factor limiting crop yields worldwide [20], and maintaining boron levels within plants under drought conditions is critical. Boron has low mobility in the phloem [21], although boron mobility in the phloem depends on plant species [3, 22]. Under water stress conditions, plant increases abscisic acid (ABA) production [23], possibly affecting photosynthetic rate in drought-stressed plants, leading to stomata closure and transpiration rate reduction. Under these conditions boron uptake and translocation, and boron movement from leaves to seed is reduced, de‐ creasing seed boron concentration [10]. Although soybean nodule growth and symbiotic N2 fixation are sensitive to drought [20,24,25], we hypothesized that drought can result in boron deficiency within the plant, impacting nodule growth, N2 fixation, and CO2 accumulation. The

© 2014 Bellaloui et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

objective of this research was to investigate the effects of foliar boron on nodules growth and nitrogen fixation rates in several soybean genotypes under water stress. To avoid the con‐ founding effects of multi-environmental factors in the field on boron application effects, the experiment was conducted under greenhouse conditions. In addition to the current research findings, the present chapter will also highlight previous and current major research findings in boron nutrition and the role of B in nodule growth and symbiotic nitrogen fixation in soybean.

#### **2. Materials and methods**

A repeated greenhouse experiment was conducted. Cultivars of maturity group (MG) III Pella, Williams 82, Hutcheson, and Forest, were used. Seeds of soybean cultivar were germinated in flat trays in vermiculite, and then uniform size seedlings at V1 stage were transplanted into 9.45 L size pots. Soil in pots was a Dundee silt loam (fine-silty, mixed, active, thermic Typic Endoqualfs) with pH 6.3, 1.1% organic matter, a cation exchange capacity of 15 cmol/kg, and soil textural fractions of 26% sand, 56% silt, and 18% clay, average B concentration was 0.72 mg kg-1. The soil contained an abundant native population of *B. japonicum*. Water stress was introduced as reported by Bellaloui [20,26]. Briefly, soil in pots were weighed and then saturated with deionized water and left to drain and weighed again to obtain the water field capacity using soil water sensors inserted in pots and measured by Soil Moisture Meter (WaterMark Company, Inc., Wisconsin, USA). Plants were divided into well watered (soil water potential between –15 to –20 kPa) (this was considered field capacity for the control plants), moderate water stress (soil water potential between -90 and -100 kPa), and severely water stressed (soil water potential between –150 to –200 kPa). Boron was foliar-applied as boric acid at a rate of 1.1 kg ha-1 once at flowering stage (R1-R2) and once at seed-fill stage (R5- R6). Combined treatments were well watered plants with no B (W-B), well watered plants with B (W+B); water stressed plants with no B (WS-B); water stressed plants with B (WS+B); severely water stressed plants with no B (SWS-B); severely water stressed plants with B (SWS+B). Samples were taken five days after the second B application for nitrate reductase assay to measure the rate nitrate reductase activity (NRA), nitrogenase, and leaf B. Mature seed were weighed at R8 (harvest maturity stage). Plants were considered fully matured when they reached R8 according to [27]. Greenhouse conditions were about 34°C ± 9°C during the day and about 28°C ± 8°C at night with a photosynthetic photon flux density (PPFD) of about 800 - 2300 µmol m-2 s-1, as measured by Quantum Meter (Spectrum Technology, Inc., Illinois, USA). The big range of light intensity reflects a bright, sunny, or cloudy day. The source of lighting was a mixture of natural light, bulb light (60 W), cool white (250 W). To avoid differences in the day-length between the two experiments, the two experiments were conducted simulta‐ neously at the same time and during the normal growing season (from April to September) for the Early Soybean Production System in the midsouth USA, and this is to be consistent with the normal photoperiod for soybean growth [10].

#### **2.1. Nitrate reductase assay**

objective of this research was to investigate the effects of foliar boron on nodules growth and nitrogen fixation rates in several soybean genotypes under water stress. To avoid the con‐ founding effects of multi-environmental factors in the field on boron application effects, the experiment was conducted under greenhouse conditions. In addition to the current research findings, the present chapter will also highlight previous and current major research findings in boron nutrition and the role of B in nodule growth and symbiotic nitrogen fixation in

A repeated greenhouse experiment was conducted. Cultivars of maturity group (MG) III Pella, Williams 82, Hutcheson, and Forest, were used. Seeds of soybean cultivar were germinated in flat trays in vermiculite, and then uniform size seedlings at V1 stage were transplanted into 9.45 L size pots. Soil in pots was a Dundee silt loam (fine-silty, mixed, active, thermic Typic Endoqualfs) with pH 6.3, 1.1% organic matter, a cation exchange capacity of 15 cmol/kg, and soil textural fractions of 26% sand, 56% silt, and 18% clay, average B concentration was 0.72 mg kg-1. The soil contained an abundant native population of *B. japonicum*. Water stress was introduced as reported by Bellaloui [20,26]. Briefly, soil in pots were weighed and then saturated with deionized water and left to drain and weighed again to obtain the water field capacity using soil water sensors inserted in pots and measured by Soil Moisture Meter (WaterMark Company, Inc., Wisconsin, USA). Plants were divided into well watered (soil water potential between –15 to –20 kPa) (this was considered field capacity for the control plants), moderate water stress (soil water potential between -90 and -100 kPa), and severely water stressed (soil water potential between –150 to –200 kPa). Boron was foliar-applied as boric acid at a rate of 1.1 kg ha-1 once at flowering stage (R1-R2) and once at seed-fill stage (R5- R6). Combined treatments were well watered plants with no B (W-B), well watered plants with B (W+B); water stressed plants with no B (WS-B); water stressed plants with B (WS+B); severely water stressed plants with no B (SWS-B); severely water stressed plants with B (SWS+B). Samples were taken five days after the second B application for nitrate reductase assay to measure the rate nitrate reductase activity (NRA), nitrogenase, and leaf B. Mature seed were weighed at R8 (harvest maturity stage). Plants were considered fully matured when they reached R8 according to [27]. Greenhouse conditions were about 34°C ± 9°C during the day and about 28°C ± 8°C at night with a photosynthetic photon flux density (PPFD) of about 800 - 2300 µmol m-2 s-1, as measured by Quantum Meter (Spectrum Technology, Inc., Illinois, USA). The big range of light intensity reflects a bright, sunny, or cloudy day. The source of lighting was a mixture of natural light, bulb light (60 W), cool white (250 W). To avoid differences in the day-length between the two experiments, the two experiments were conducted simulta‐ neously at the same time and during the normal growing season (from April to September) for the Early Soybean Production System in the midsouth USA, and this is to be consistent

soybean.

**2. Materials and methods**

238 Advances in Biology and Ecology of Nitrogen Fixation

with the normal photoperiod for soybean growth [10].

The rate of nitrate reductase activity (NRA) was determined according to [28, 29]. Briefly, NRA was measured in the fully expanded leaves and nodules. Nodules were gently and carefully separated from roots and placed in NRA assay buffer solution. A fresh leaf sample of about 0.3 g was placed in 10 mL of potassium phosphate buffer at a concentration of 100 mM, pH 7.5, containing 1% (v/v) 1-propanol, in the flask. The incubation solution was vacuum-filtered for 1 min, and then flashed with nitrogen gas for 30 s, and then incubated at 30°C. A samples of 0.5 mL was taken at regular intervals (0, 60, 120, 180, and 300 min) for nitrite measurement. Samples were extracted with 5 mL of deionized water and reacted with 1.0 mL of 1% (w/v) sulfanilamide in 10% v/v HCl and 1.0 mL of *N*-naphthyl-(1)-ethylenediamine dihydrochloride (0.1%). Nitrite concentration in samples was measured by reading the absorbance at 540 nm after 30 minutes using a Beckman Coulter DU 800 spectro- photometer (Fullerton, CA). A standard curve was produced using KNO2 as a source of NO2 in the tested samples according to (Bellaloui et al., 2006). To measure the enzyme activity where there was no limiting concentration of NO3 in the incubation culture solution (potential nitrate reductae activity, PNRA), exogenous NO3 was added at a concentration of 10 mM as KNO3.

#### **2.2. Acetylene reduction assay**

Destructive method for acetylene reduction assay was used. Three plants from each replicate were harvested five days after the second B application (at seed-fill stage). Nitrogenase activity was assayed using the acetylene reduction assay as described elsewhere [29-31]. Roots with nodules intact were excised and incubated in 60 mL plastic syringes. Roots from each replicate and from each treatment were placed in the syringes in the Mason jars and sealed. A 10% volume of air was then removed and replaced with an equal volume of acetylene. After 1 h of incubation at room temperature, duplicate 1.0 mL gas samples were removed and analyzed by gas chromatography for ethylene formation and carbon dioxide evolution. The gas chromatography (Agilent HP6960, Agilent Technologies, Wilmington, DE) was equipped with manual injector, injector loop, and sample splitter. A flame ionization detector (FID) and a thermal conductivity detector (TCD) were used. Using the sample loop and splitter, 0.25 mL of gas was directed into a 30 m length × 0.53 mm i.d. alumina megabore column (115-3532) connected to the FID, and 0.25 mL of sample was injected into a HP-PLOT D column (30 m length × 0.53 mm i.d. megabore with 40 µm film; 1905D-Q04) connected to the TCD using helium as a carrier gas. Chromatographs were integrated using Chem Station software. Standard curves for ethylene and carbon dioxide were updated and produced for each day. Samples having <9% acetylene were not used in the analysis.. Nodules were carefully removed and counted, and then oven-dried at 60°C for 4 - 5 days.

#### **2.3. Boron determination**

The concentration of total B was measured in the fully expanded leaves after the second foliar B application, and in seeds at harvest maturity stage. Boron concentration was determined according to Azomethine—H method [19,32-34]. Briefly, 1 g of dry sample was placed in a porcelain crucible for ashing at 500°C for 8 hr. Samples then were extracted with 20 mL of 2 M HCl at 90°C for 10 min and azomethine-H solution containing 0.45% before the analysis (John et al., 1975) with a buffer solution contained 25% ammonium acetate, 1.5% EDTA, and 12.5% acetic acid. The concentration of B in the samples were determined spectrophotometri‐ cally by using color development after 45 minutes. Samples were read at 420 nm using a Beckman Coulter DU 800 spectrophotometer (Fullerton, California). Boron analysis in soil was conducted using Inductively Coupled Plasma spectrometry (ICP) using Thermo Elemental, Thermo Jarrell-Ash model 61E ICP, USA [10].

#### **2.4. Analysis of δ15N (15N/14N ratio) and δ13C (13C/12C ratio) using natural abundance**

Natural abundance of δ 15N and 13C isotopes was determined using about 0.9 mg of ground seeds. Isotopic analysis was conducted using a Thermo FinniGlyn Delta Plus Advantage Mass Spectrometer with a FinniGlyn ConFlo III, and Isomass Elemetal Analyzer (Bremen, Germany) according to [26, 35,36]. Isodat software version 2.38 was used to obtain Delta values [26]. The elemental combustion system was Costech ECS 4010 with an autosampler (Bremen, Germany).

#### **2.5. Determination of seed sucrose**

Seed sucrose concentration was measured in mature seeds. Sucrose concentration was measured according to [37,38] using an AD 7200 diode array feed analyzer (Perten, Springfield, IL). Briefly, about 25 g of seed were ground using a Laboratory Mill 3600 (Perten, Springfield, IL). Initial calibration equations were developed by the Department of Agronomy and Plant Genetics, University of Minnesota St Paul, MN using Thermo Galactic Grams PLS IQ software, developed by Perten company (Perten, Springfield, IL). Analyses of sugars were performed based on a seed dry matter basis [26, 37, 39].

#### **2.6. Seed glucose determination**

Glucose concentration in mature seeds was measured enzymatically using Glucose (HK) Assay Kit from Sigma, USA, Product Code GAHK-20, 2012) [40]. In this reaction, glucose is phosphorylated by adenosine triphosphate (ATP) in a reaction catalyzed by hexokinase. Glucose-6-phosphate (G6P) produced is then oxidized to form 6-phosphogluconate in the presence of oxidized nicotinamide adenine dinucleotide (NAD) in a reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). During this oxidation, an equimolar amount of NAD is reduced to NADH, and the increase in absorbance at 340 nm is directly proportional to glucose concentration in the sample. Mature seed samples were ground using a Laboratory Mill 3600 (Perten, Springfield, IL). A dry, ground sample of 0.1 mg was extracted with deionized water. The extraction procedure of glucose from seeds was conducted according to [26], and as instructed by Glucose (HK) Assay Kit from Sigma. The concentration of glucose was measured spectrophotometrically by reading the samples at 340nm using a Beckman Coulter DU 800 spectrophotometer (Fullerton, CA). The concentration of glucose was ex‐ pressed as mg g dwt-1.

#### **2.7. Seed fructose determination**

M HCl at 90°C for 10 min and azomethine-H solution containing 0.45% before the analysis (John et al., 1975) with a buffer solution contained 25% ammonium acetate, 1.5% EDTA, and 12.5% acetic acid. The concentration of B in the samples were determined spectrophotometri‐ cally by using color development after 45 minutes. Samples were read at 420 nm using a Beckman Coulter DU 800 spectrophotometer (Fullerton, California). Boron analysis in soil was conducted using Inductively Coupled Plasma spectrometry (ICP) using Thermo Elemental,

**2.4. Analysis of δ15N (15N/14N ratio) and δ13C (13C/12C ratio) using natural abundance**

Natural abundance of δ 15N and 13C isotopes was determined using about 0.9 mg of ground seeds. Isotopic analysis was conducted using a Thermo FinniGlyn Delta Plus Advantage Mass Spectrometer with a FinniGlyn ConFlo III, and Isomass Elemetal Analyzer (Bremen, Germany) according to [26, 35,36]. Isodat software version 2.38 was used to obtain Delta values [26]. The elemental combustion system was Costech ECS 4010 with an autosampler (Bremen, Germany).

Seed sucrose concentration was measured in mature seeds. Sucrose concentration was measured according to [37,38] using an AD 7200 diode array feed analyzer (Perten, Springfield, IL). Briefly, about 25 g of seed were ground using a Laboratory Mill 3600 (Perten, Springfield, IL). Initial calibration equations were developed by the Department of Agronomy and Plant Genetics, University of Minnesota St Paul, MN using Thermo Galactic Grams PLS IQ software, developed by Perten company (Perten, Springfield, IL). Analyses of sugars were performed

Glucose concentration in mature seeds was measured enzymatically using Glucose (HK) Assay Kit from Sigma, USA, Product Code GAHK-20, 2012) [40]. In this reaction, glucose is phosphorylated by adenosine triphosphate (ATP) in a reaction catalyzed by hexokinase. Glucose-6-phosphate (G6P) produced is then oxidized to form 6-phosphogluconate in the presence of oxidized nicotinamide adenine dinucleotide (NAD) in a reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). During this oxidation, an equimolar amount of NAD is reduced to NADH, and the increase in absorbance at 340 nm is directly proportional to glucose concentration in the sample. Mature seed samples were ground using a Laboratory Mill 3600 (Perten, Springfield, IL). A dry, ground sample of 0.1 mg was extracted with deionized water. The extraction procedure of glucose from seeds was conducted according to [26], and as instructed by Glucose (HK) Assay Kit from Sigma. The concentration of glucose was measured spectrophotometrically by reading the samples at 340nm using a Beckman Coulter DU 800 spectrophotometer (Fullerton, CA). The concentration of glucose was ex‐

Thermo Jarrell-Ash model 61E ICP, USA [10].

240 Advances in Biology and Ecology of Nitrogen Fixation

**2.5. Determination of seed sucrose**

based on a seed dry matter basis [26, 37, 39].

**2.6. Seed glucose determination**

pressed as mg g dwt-1.

Fructose concentration is mature seeds was determined enzymatically according to Fructose Assay Kit from Sigma, USA, Product Code FA-20, 2012 [41]. In this reaction, fructose is phosphorylated by ATP in a reaction catalyzed by hexokinase, and the produced fructose 6 phosphate is then converted to G6P by phosphoglucose isomerase (PGI). The oxidation of G6P to 6-phosphogluconate takes place in the presence of NAD in the reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). An equimolar amount of NAD is reduced to NADH, and the consequent increase in absorbance at 340 nm is directly proportional to fructose concentration in a sample. A sample of 0.1 mg was extracted according to Fructose Assay Kit from Sigma, and is detailed in Bellaloui et al (2013) as instructed by Fructose Assay Kit from Sigma. The concentration of fructose in samples were measured spectrophotometri‐ cally by reading the samples at absorbance of 340nm using a Beckman Coulter DU 800 spectrophotometer (Fullerton, CA). The concentration of fructose was expressed as mg g dwt-1.

#### **2.8. Experimental design and statistical analysis**

Treatments were arranged in a split plot design with irrigation as a main block and B treatment as sub-plot. Four replicates were used, and each replicate consisted of a pot containing three plants. Proc Mixed was used for data analysis of variance in SAS [42]. Means were separated by Fisher's least significant difference test at the 5% level of probability using Proc GLM analysis in SAS [42]. Since there were no interactions between the two experiments, the data were pooled and combined.

#### **3. Results and discussion**

Analysis of variance showed that boron application (T) and irrigation (IR) were significant for grain weight, nodule mass, nitrogen fixation (NF), nitrate reductase activity (NRA), N, B, and natural abundance of δ<sup>15</sup>N and δ13C (Table 1), and sugars (Table 2). Cultivar (CV) was signif‐ icant for some parameters and not significant for others, indicating differences in cultivar responses to the B application and water stress. Both B application and IR significantly interacted (T×IR and E×T×IR interactions) for these parameters, indicating that B effects on these parameters depended on IR (watered or water stressed conditions). There were no significant interactions between Experiment (E) and T or IR, indicating that the effect of B application or IR had similar effect in each experiment (Table 1). Therefore, the data were pooled and combined [26].

#### **3.1. Effect of B and water stress on grain weight, nodule mass and nodule number**

Foliar B application to watered plants (W+B) resulted in significant increase (P≤0.05) in grain weight, nodule mass, and nodule number (Table 3). For example, in Pella cultivar the increase of grain weight, nodule mass, and nodule number was 18.7%, 38.5%, and 33.3 %, respectively. These parameters were different between cultivars and each cultivar responded differently to foliar B (Table 3). Previous research indicated that B plays a major role for plant growth and development [1, 2] and crop quality [4,19]. It was shown that foliar B improved seed set, seed yield, and seed quality of alfalfa [19] and sugar beet [4], and altered seed composition in soybean [10]. Previous research showed that B is an essential nutrient for the development of nitrogen-fixing root nodules in pea (*Pisum sativum*) [9]. A lower level of infection of the host plants with *Rhizobium* was noticed in plants grown in B-deficient medium compared to plants supplied with adequate B [9]. It was shown that that little or no ability to fix N2 under Bdeficient plants [11]. Recently, it was found that FB increased nodule weight under irrigated greenhouse conditions [43]. The current results showed that, even though B concentration in leaves was above the critical level (20 mg B kg-1, critical level of B in leaves for normal plant growth) [44], FB resulted in a positive effect on seed and nodule weights, agreeing with previous research of those of [7, 43, 45]. Our results showed that foliar B increased grain weight, nodule mass and number due to the stimulatory effects of B on growth and development [9,11] and nodule improvement.

#### **3.2. Effect of B and water stress on nitrogen fixation and nitrogen assimilation**

Foliar application of B resulted in higher rates of nitrogen fixation (increase of nitrogenase), root respiration, and nitrogen assimilation (increase of nitrate reductase activity, NRA), (Table 4). Foliar B application to moderately water stressed plants (Table 4) increased grain weight, nodule number and mass, nitrogen fixation and assimilation. However, foliar B application to severely water stress plants did not result in an increase in these parameters because of the damaging effects of water stress to nitrogen metabolism enzymes, especially nitorgenase and nitrate reductase (Table 5). Researchers reported that B is an essential micronutrient for the development of nitrogen-fixing root nodules [11], and plants grown in B-deficient medium showed lower infection of the host plants with Rhizobium compared to plants supplied with adequate B [11]. It was reported that nodules showed little or no ability to fix N2 in B-deficient plants, leading to N deficiency and necrosis of nodulated pea plants [9]. This indicated that nitrogen fixation in soybean was sensitive to B deficiency [9,12], and B deficiency can result in the reduction in early nodullin protein (ENOD2) in nodule parenchyma cells and malfunction of oxygen diffusion barrier [13]. It was hypothesized that B protects nitrogenase against oxygen damage by influencing membrane integrity and function [13] and may interact with membrane glycoproteins and glycolipids to maintain the proper conformation in nitrogen-fixing cells [5]. Although B has been shown to be essential for nodule growth and development, there is no convincing evidence that there is a direct effect of B on nitrogen metabolism [2,13,46,]. Nitrate assimilation, reflected by the key enzyme in nitrogen assimilation, was higher in W+B plants, indicating that B enhanced nitrate assimilation. The stimulatory effects of nitrogen assimilation by B was also reported by others (Bellaloui et al., 2011 AJPS. This is because nitrogen metab‐ olism in legumes is both a result of both symbiotic N2 fixation and mineral N assimilation processes. During this process, atmospheric N2 is fixed by the enzyme nitrogenase in the bacteroids of nodules [47], and nitrate reduction (assimilation) is catalyzed by the enzyme nitrate reductase (NR). Both NR and nitrogenase enzymes coexist in nodules competing for reductant [48]. It appears that B may stimulate de novo synthesis and making nitrate (enzyme substrate) available for the enzyme nitrate reductase. Adding 0.5 mM B to the buffer solution increased NRA by 30% in WS+B compared with WS-B, and adding 10 mM NO3 to the buffer solution increased NRA by 55% and 40% in leaves and nodules, respectively (data not shown). In SWS plants, adding B or NO3 did not enhance NRA (data not shown). This indicated that both B and NO3 stimulated NR enzyme somehow, maybe by facilitating nitrate availability in the cytoplasm to NR for reduction or enhancing nitrate translocation from the vacuoles to the cytoplasm, leading to higher NRA activity. This hypothesis was supported by the effect of B ion uptake [2,16] and the direct or indirect effects of B on the plasma membrane bound H+ ATPase (plasmalemma H+-ATPase activity) [7,17], cell wall structure and membrane integrity [2,7]. Our results are supported by [2] in that B may have an indirect influence on nitrate uptake and assimilation, and enhance NRA by inducing nitrate availability and increasing protein de novo synthesis as a result of nitrate absorption [49]. This observation is supported by [13] who found that adequate level of B increased NRA and decreased nitrate in xylem sap compared to deficiency level. The relationship between nitrogen fixation and nitrogen assimilation and how this relationship is influenced by foliar B and its impact on seed protein and oil and sugars is still not well established.

foliar B (Table 3). Previous research indicated that B plays a major role for plant growth and development [1, 2] and crop quality [4,19]. It was shown that foliar B improved seed set, seed yield, and seed quality of alfalfa [19] and sugar beet [4], and altered seed composition in soybean [10]. Previous research showed that B is an essential nutrient for the development of nitrogen-fixing root nodules in pea (*Pisum sativum*) [9]. A lower level of infection of the host plants with *Rhizobium* was noticed in plants grown in B-deficient medium compared to plants supplied with adequate B [9]. It was shown that that little or no ability to fix N2 under Bdeficient plants [11]. Recently, it was found that FB increased nodule weight under irrigated greenhouse conditions [43]. The current results showed that, even though B concentration in leaves was above the critical level (20 mg B kg-1, critical level of B in leaves for normal plant growth) [44], FB resulted in a positive effect on seed and nodule weights, agreeing with previous research of those of [7, 43, 45]. Our results showed that foliar B increased grain weight, nodule mass and number due to the stimulatory effects of B on growth and development [9,11]

**3.2. Effect of B and water stress on nitrogen fixation and nitrogen assimilation**

adequate B [11]. It was reported that nodules showed little or no ability to fix N2

Foliar application of B resulted in higher rates of nitrogen fixation (increase of nitrogenase), root respiration, and nitrogen assimilation (increase of nitrate reductase activity, NRA), (Table 4). Foliar B application to moderately water stressed plants (Table 4) increased grain weight, nodule number and mass, nitrogen fixation and assimilation. However, foliar B application to severely water stress plants did not result in an increase in these parameters because of the damaging effects of water stress to nitrogen metabolism enzymes, especially nitorgenase and nitrate reductase (Table 5). Researchers reported that B is an essential micronutrient for the development of nitrogen-fixing root nodules [11], and plants grown in B-deficient medium showed lower infection of the host plants with Rhizobium compared to plants supplied with

plants, leading to N deficiency and necrosis of nodulated pea plants [9]. This indicated that nitrogen fixation in soybean was sensitive to B deficiency [9,12], and B deficiency can result in the reduction in early nodullin protein (ENOD2) in nodule parenchyma cells and malfunction of oxygen diffusion barrier [13]. It was hypothesized that B protects nitrogenase against oxygen damage by influencing membrane integrity and function [13] and may interact with membrane glycoproteins and glycolipids to maintain the proper conformation in nitrogen-fixing cells [5]. Although B has been shown to be essential for nodule growth and development, there is no convincing evidence that there is a direct effect of B on nitrogen metabolism [2,13,46,]. Nitrate assimilation, reflected by the key enzyme in nitrogen assimilation, was higher in W+B plants, indicating that B enhanced nitrate assimilation. The stimulatory effects of nitrogen assimilation by B was also reported by others (Bellaloui et al., 2011 AJPS. This is because nitrogen metab‐ olism in legumes is both a result of both symbiotic N2 fixation and mineral N assimilation processes. During this process, atmospheric N2 is fixed by the enzyme nitrogenase in the bacteroids of nodules [47], and nitrate reduction (assimilation) is catalyzed by the enzyme nitrate reductase (NR). Both NR and nitrogenase enzymes coexist in nodules competing for reductant [48]. It appears that B may stimulate de novo synthesis and making nitrate (enzyme substrate) available for the enzyme nitrate reductase. Adding 0.5 mM B to the buffer solution

in B-deficient

and nodule improvement.

242 Advances in Biology and Ecology of Nitrogen Fixation


\* Significance at *P* ≤ 0.05; \*\* Significance at *P* ≤ 0.01; \*\*\* Significance at *P* ≤ 0.001.

**Table 1.** Analysis of variance of the effects of foliar boron on seed weight (100 seed weight, g), nodule mass (mg plant-1), nodule number plant-1, [nitrogen fixation (acetylene reduction assay (ARA), μmol of C2H4 plant-1 h-1)], leaf nitrate reductase activity (NRA, µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1), boron (B, mg kg-1)) and nitrogen (N, %) in leaves and seeds, and in δ15N and in δ 13C isotope values in seeds in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered and water stressed conditions (WS) with and without foliar boron (B) treatments (T) under greenhouse conditions *<sup>a</sup>* .


\* Significance at *P* ≤ 0.05; \*\* Significance at *P* ≤ 0.01; \*\*\* Significance at *P* ≤ 0.001.

**Table 2.** Analysis of variance of the effects of foliar boron on sugars (mg g-1 dwt) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered and water stressed conditions (IR) with and without foliar boron (B) treatments (T) under greenhouse conditions *<sup>a</sup>* .


*a* Soybean plants were grown at field capacity at -15 to -20 kPa [10]. Soybeans were grown under greenhouse condi‐ tions similar to those in Bellaloui et al. (2011). Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers' test. W 82=Williams 82.

**Table 3.** Effect of foliar boron on soybean seed weight (100 seed weight, g), nodule mass (mg plant-1), nodule number plant-1, ARA (μmol of C2H4 plant-1 h-1), root respiration (mmol of CO2 evolved/g of root/h), leaf NRA (µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered conditions without boron (W-B) and with boron (W+B) under greenhouse conditions *<sup>a</sup>* .


**Source of variability Glucose Fructose Sucrose Raffinose Stachyose** Experiment (E) NS NS NS NS NS Treatment (T) \*\* \* \* \* \*\*\* Irrigation (IR) \*\* \* \* \*\* \*\*\* Cultivar (CV) \* \* \* NS NS E×T NS NS NS NS NS E×IR NS NS NS NS NS E×CV NS NS NS NS NS T×IR \* \* \* \*\* \* T×CV \*\* \* \* \* \* CV×IR \* \*\* \* \* \* E×T×IR×CV \* \* \* \*\* \*

**Table 2.** Analysis of variance of the effects of foliar boron on sugars (mg g-1 dwt) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered and water stressed conditions (IR) with

**Watered soybean**

Pella W-B 16 a 65 a 33 a 11.6 a 7.5 a 5.6 ab 4.7 ab W 82 15 a 68 a 28 b 10.5 a 7.8 a 4.9 b 5.2 a

on 14 b 61 b 25 b 10.1 a 6.8 b 5.7 ab 3.8 b Forrest 14 b 64 a 17 c 11.4 a 6.4 b 6.3 a 4.2 ab Pella 19 a 90 a 44 a 15.4 a 9.7 b 7.5 ab 6.4 a W 82 W+B 18 a 86 ab 38 abc 14.7 ab 10.5 ab 6.8 b 7.8 a

on 16 b 84 b 36 c 13.2 b 10.6 ab 7.4 ab 5.2 b Forrest 17 b 86 ab 38 abc 13.6 b 11.5 a 8.3 a 5.8 b

 Soybean plants were grown at field capacity at -15 to -20 kPa [10]. Soybeans were grown under greenhouse condi‐ tions similar to those in Bellaloui et al. (2011). Values within columns and within each B treatment sharing a letter are

**Table 3.** Effect of foliar boron on soybean seed weight (100 seed weight, g), nodule mass (mg plant-1), nodule number plant-1, ARA (μmol of C2H4 plant-1 h-1), root respiration (mmol of CO2 evolved/g of root/h), leaf NRA (µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered conditions without boron (W-B) and with boron (W+B) under greenhouse conditions *<sup>a</sup>*

**ARA (NF) (μmol of C2H4 plant-1 h-1)**

**Nodule number plant-1**

.

R**oot respiration (mmol of CO2 evolved/g of root/h)**

**Leaf NRA (µmol NO2 g-1 h-1)**

**Nodule NRA (µmol NO2 g-1h-1)**

.

\* Significance at *P* ≤ 0.05; \*\* Significance at *P* ≤ 0.01; \*\*\* Significance at *P* ≤ 0.001.

and without foliar boron (B) treatments (T) under greenhouse conditions *<sup>a</sup>*

**Nodule mass (mg dwt plant-1)**

not significantly different (P>0.05) using Fishers' test. W 82=Williams 82.

**Variety Boron Grain**

Hutches

Hutches

*a*

**weight (100 seed weight, g)**

244 Advances in Biology and Ecology of Nitrogen Fixation

a Soybean plants were grown under water stress (WS) (-90 to -100 kPa soil water potential). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B treat‐ ment sharing a letter are not significantly different (P>0.05) using Fishers' test. W 82=Williams 82.

**Table 4.** Effect of foliar boron on soybean seed weight (g), nodule mass (mg plant-1), nodule number plant-1, ARA ((μmol of C2H4 plant-1 h-1), root respiration (mmol of CO2 evolved/g of root/h), leaf NRA (µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under water stress conditions without boron (WS-B) and with boron (WS+B) under greenhouse conditions a .


a Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers' test. W 82=Williams 82.

**Table 5.** Effect of foliar boron on soybean seed weight (g), nodule mass (mg plant-1), nodule number plant-1, nitrogen fixation ( ARA, μmol of C2H4 plant-1 h-1), root respiration (mmol of CO2 evolved/g of root/h), leaf NRA (µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under severe water stress conditions without boron (SWS-B) and with boron (SWS+B) under greenhouse conditions a .

Foliar boron application increased B in leaves and seed in watered plants (Figure 1). No significant B concentration differences were observed between leaves and seeds B in each watered treatment in each cultivar, indicating that B movement from leaves to seeds was not limited. In severely water-stressed plants, application of foliar B did not significantly increase B in leaves, and B movement from leaves to seed was limited, indicated by the large accumu‐ lation of B in leaves and small accumulation of B in seeds. Similar trend of N in leaves and seed was noticed (Figure 2), indicating a close relationship between B and N.

#### **3.3. Effects of B and water stress on seed sugars**

Since B plays an important role in carbohydrate mobility and since carbohydrates are a source of reducing power in nitrogen assimilation, sugar profiling was also investigated. Our research demonstrated that foliar B application resulted in higher sucrose, glucose, and fructose under irrigated conditions, but under severe water stress these mono and disaccharides sugars decreased, but stachyose and raffinose increased (Table 6,7,8).

This indicated that there was a redistribution of sugars under severe water stress, and this shift may provide plants with an adaptive mechanism to tolerate the stress.

Foliar boron resulted in higher seed sucrose, glucose, and fructose concentrations in W+B plants, indicating B involvement in sugar metabolism and synthesis. The involvement of B in sugar synthesis and distribution is not understood, but B involvement in sugar movement and metabolism was previously reported [2,3,50]. The decrease of sucrose, glucose, and fructose in SWS-B and SWS+B plants compared with W+B and W-B indicated that B enhanced accu‐ mulation of sucrose, glucose, and fructose concentrations may be due to B role in sugar movement within the plants, and that severe water stress limited sugars movement due reduction of B uptake and stomatal conductance. The increase of seed stachyose concentration in seeds of SWS-B and SWS+B plants indicated that severe water stress affects the distribution of sugar fractions, in our case the increase of stachyose and raffinose and decrease of sucrose, glucose, and fructose concentrations. The increase of stachyose under water stress may indicate the role of stachyose in plant tolerance to biotic and abiotic stress [14,15]. It was also reported that raffinose and galactinol levels may play an important role in plant tolerance to biotic and abiotic stress [14,15], and the accumulation of galactinol and raffinose may protect the plant from drought [51], and the activity of sucrose synthase, the main enzyme involved in sucrose hydrolysis in nodules, was significantly inhibited under drought conditions [52,53]. The biological functions of raffinose and stachyose are not clear [54], but previous research reported that oligosaccharides (sucrose, raffinose, and stachyose) are related to seed quality [55] and the acquisition of desiccation tolerance during seed development and maturation.

Soybean seed sugars are important to soybean seed industry because they determine the quality of seeds beside protein and oil. This is because soybean seed with high raffinose and stachyose concentrations are undesirable and have negative effects on the nutritive value of soymeal and seed consumed by human. Stachyose and raffinose are indigestible by humans and animals, especially monogastric animal such as chicken and pigs, causing flatulence or diarrhea [56]. On the other hand, low raffinose and stachyose levels in soybean seed are desirable [57]. High level of seed sucrose, glucose, and fructose are desirable because it improves taste and flavor of tofu, soymilk, and natto [58]. Currently, soybean cultivars with improved sugar profiles have been released to the market [58], and breeding for desirable sugars in soybean or agricultural practices to improve seed sugars are needed.

Foliar boron application increased B in leaves and seed in watered plants (Figure 1). No significant B concentration differences were observed between leaves and seeds B in each watered treatment in each cultivar, indicating that B movement from leaves to seeds was not limited. In severely water-stressed plants, application of foliar B did not significantly increase B in leaves, and B movement from leaves to seed was limited, indicated by the large accumu‐ lation of B in leaves and small accumulation of B in seeds. Similar trend of N in leaves and

Since B plays an important role in carbohydrate mobility and since carbohydrates are a source of reducing power in nitrogen assimilation, sugar profiling was also investigated. Our research demonstrated that foliar B application resulted in higher sucrose, glucose, and fructose under irrigated conditions, but under severe water stress these mono and disaccharides sugars

This indicated that there was a redistribution of sugars under severe water stress, and this shift

Foliar boron resulted in higher seed sucrose, glucose, and fructose concentrations in W+B plants, indicating B involvement in sugar metabolism and synthesis. The involvement of B in sugar synthesis and distribution is not understood, but B involvement in sugar movement and metabolism was previously reported [2,3,50]. The decrease of sucrose, glucose, and fructose in SWS-B and SWS+B plants compared with W+B and W-B indicated that B enhanced accu‐ mulation of sucrose, glucose, and fructose concentrations may be due to B role in sugar movement within the plants, and that severe water stress limited sugars movement due reduction of B uptake and stomatal conductance. The increase of seed stachyose concentration in seeds of SWS-B and SWS+B plants indicated that severe water stress affects the distribution of sugar fractions, in our case the increase of stachyose and raffinose and decrease of sucrose, glucose, and fructose concentrations. The increase of stachyose under water stress may indicate the role of stachyose in plant tolerance to biotic and abiotic stress [14,15]. It was also reported that raffinose and galactinol levels may play an important role in plant tolerance to biotic and abiotic stress [14,15], and the accumulation of galactinol and raffinose may protect the plant from drought [51], and the activity of sucrose synthase, the main enzyme involved in sucrose hydrolysis in nodules, was significantly inhibited under drought conditions [52,53]. The biological functions of raffinose and stachyose are not clear [54], but previous research reported that oligosaccharides (sucrose, raffinose, and stachyose) are related to seed quality [55] and

the acquisition of desiccation tolerance during seed development and maturation.

Soybean seed sugars are important to soybean seed industry because they determine the quality of seeds beside protein and oil. This is because soybean seed with high raffinose and stachyose concentrations are undesirable and have negative effects on the nutritive value of soymeal and seed consumed by human. Stachyose and raffinose are indigestible by humans and animals, especially monogastric animal such as chicken and pigs, causing flatulence or diarrhea [56]. On the other hand, low raffinose and stachyose levels in soybean seed are desirable [57]. High level of seed sucrose, glucose, and fructose are desirable because it

seed was noticed (Figure 2), indicating a close relationship between B and N.

**3.3. Effects of B and water stress on seed sugars**

246 Advances in Biology and Ecology of Nitrogen Fixation

decreased, but stachyose and raffinose increased (Table 6,7,8).

may provide plants with an adaptive mechanism to tolerate the stress.


a Soybean plants were grown at field capacity at -15 to -20 kPa according to Bellaloui et al., (2011). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within in each B treatment sharing a letter are not significantly different (P>0.05) using Fishers' test. W 82=Williams 82.

**Table 6.** Effect of foliar boron on soybean seed sugars in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered conditions without boron (W-B) and with boron (W+B) under greenhouse conditions a .


a Soybean plants were grown under water stress (WS) (-90 to -100 kPa soil water potential). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers' test. W 82=Williams 82.

**Table 7.** Effect of foliar boron on soybean seed sugars in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under water stress conditions without boron (WS-B) and with boron (WS+B) under greenhouse conditions a .


a Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers' test. W 82=Williams 82.

**Table 8.** Effect of foliar boron on soybean seed sugars in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under severe water stress conditions without boron (SWS-B) and with boron (SWS+B) under greenhouse conditions a .

#### **3.4. Effect of B and water stress on δ15N (15N/14N ratio) and δ13C (13C/12C ratio)**

Foliar B did not result in changes in 15N/14N or 13C/12C ratios, but significant differences in these ratios were observed between irrigated and non-irrigated soybean with or without foliar B (Figure 3). The alteration of 15N/14N by increasing 15N (derived from soil nitrogen that is used for nitrate assimilation) and decreasing 14N (derived from atmospheric nitrogen that is used for nitrogen fixation) indicated that the source of nitrogen use changed, and plants favored N from soil over atmospheric nitrogen, indicating that nitrogenase is more sensitive than nitrate reductase under water stress. The mechanisms of this shift are not understood, but one possible explanation is that the shift in 15N/14N may reflect a possible mechanism to compensate for the inhibition of nitrogen fixation under water stress conditions. Previous research indicated that δ15N values in the xylem and plant tissues were associated with acquired N, and changed with N metabolism [59]. The increase in δ13C or higher 13C/12C ratio (less negative) in seed under severe water stress conditions indicated that the source of carbon fixation used was shifted. Previous research reported that that the δ13C value in plant tissues can be affected by water supply [60], plant physiology [61], and mycorrhizal infection [62]. The level of δ13C was dependent on the environmental factors and their association with plant gas exchange, stomatal conductance, and CO2 fixation [63]. It was found that drought stress leads to stomatal closure and 13C fixation increase, resulting in less discrimination against δ13C [64,65]. Previous research indicated that the the shift in 13C/12C ratio was a result of a shift in carbon fixation metabolism from ribulose bisphosphate (RuBP) carboxylase pathway to phosphoenolpyru‐ vate carboxylase (PEP). This shift resulted in δ13C enrichment [60]. It was found that in C3 species, to which soybean belongs, carbon isotope composition changes among and between

**Variety Boron Glucose**

248 Advances in Biology and Ecology of Nitrogen Fixation

under greenhouse conditions a

a

**(mg g-1)**

.

**Fructose (mg g-1)**

Pella SWS-B 0.65 a 0.54 a 17.5 a 8.5 b 73.5 a W 82 0.53 b 0.43 b 14.3 b 9.5 a 65.4 b Hutcheson 0.51 b 0.47 b 15.3 ab 8.3 b 73.7 a Forrest 0.48 c 0.52 a 11.6 c 9.5 a 65.7 b Pella 0.59 a 0.53 a 19.5 a 8.5 b 62.1 b W 82 SWS+B 0.48 c 0.47 b 13.2 b 9.5 a 73.6 a Hutcheson 0.52 b 0.51 a 17.6 a 8.7 b 68.5 ab Forrest 0.51 b 0.48 b 14.2 b 7.2 c 68.5 ab

 Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B

**Table 8.** Effect of foliar boron on soybean seed sugars in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under severe water stress conditions without boron (SWS-B) and with boron (SWS+B)

Foliar B did not result in changes in 15N/14N or 13C/12C ratios, but significant differences in these ratios were observed between irrigated and non-irrigated soybean with or without foliar B (Figure 3). The alteration of 15N/14N by increasing 15N (derived from soil nitrogen that is used for nitrate assimilation) and decreasing 14N (derived from atmospheric nitrogen that is used for nitrogen fixation) indicated that the source of nitrogen use changed, and plants favored N from soil over atmospheric nitrogen, indicating that nitrogenase is more sensitive than nitrate reductase under water stress. The mechanisms of this shift are not understood, but one possible explanation is that the shift in 15N/14N may reflect a possible mechanism to compensate for the inhibition of nitrogen fixation under water stress conditions. Previous research indicated that δ15N values in the xylem and plant tissues were associated with acquired N, and changed with N metabolism [59]. The increase in δ13C or higher 13C/12C ratio (less negative) in seed under severe water stress conditions indicated that the source of carbon fixation used was shifted. Previous research reported that that the δ13C value in plant tissues can be affected by water supply [60], plant physiology [61], and mycorrhizal infection [62]. The level of δ13C was dependent on the environmental factors and their association with plant gas exchange, stomatal conductance, and CO2 fixation [63]. It was found that drought stress leads to stomatal closure and 13C fixation increase, resulting in less discrimination against δ13C [64,65]. Previous research indicated that the the shift in 13C/12C ratio was a result of a shift in carbon fixation metabolism from ribulose bisphosphate (RuBP) carboxylase pathway to phosphoenolpyru‐ vate carboxylase (PEP). This shift resulted in δ13C enrichment [60]. It was found that in C3 species, to which soybean belongs, carbon isotope composition changes among and between

treatment sharing a letter are not significantly different (P>0.05) using Fishers' test. W 82=Williams 82.

**3.4. Effect of B and water stress on δ15N (15N/14N ratio) and δ13C (13C/12C ratio)**

**Sucrose (mg g-1)** **Raffinose (mg g-1)**

**Stachyose (mg g-1)**

Figure 1. Effects of foliar boron application (1.1 kg B ha-1) on boron concentration in leaves and seed in soybean genotypes in watered (A,B) and severe water stressed (C,D) soybean genotypes. Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. **Figure 1.** Effects of foliar boron application (1.1 kg B ha-1) on boron concentration in leaves and seed in soybean gen‐ otypes in watered (A,B) and severe water stressed (C,D) soybean genotypes. Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10].

genotypes correlated with water use efficiency, and the stable isotope 13C would be discrimi‐ nated against during photosynthesis, leading to a smaller 13C to 12C ratio [66]. The enrichment of 13C may be due closure of stomatal conductance under severe water stress, leading to 13C fixation increase and less 13C discrimination [67,68]. Our current results are in agreement with previous reports that environmental stresses, including drought, can alter δ13C due to the drought effects on the balance between stomatal conductance and carboxylation [67,68,69].

Figure 2. Effects of foliar boron application (1.1 kg B ha-1) on nitrogen percentage in leaves and seed in soybean genotypes in watered (A,B) and severe water stressed (C,D) soybean genotypes. Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Soybean seed sugars are important to soybean seed industry because they determine the quality of seeds beside protein and oil. **Figure 2.** Effects of foliar boron application (1.1 kg B ha-1) on nitrogen percentage in leaves and seed in soybean geno‐ types in watered (A,B) and severe water stressed (C,D) soybean genotypes. Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10].

During carbon fixation by photosynthesis, the naturally occurring stable isotope 13C is discriminated against, and plants would have a smaller 13C to 12C ratio than 13C to 12C ratio in fixed CO2 of the air, suggesting a possible use of this technique to select for water use efficiency (Farquhar et al., 1989*)*. Our results demonstrated that δ 15N and δ13C values changed and enrichment occurred under water stress conditions, suggesting that both nitrogen and carbon metabolism pathways were affected during water stress, impacting seed production and seed quality. This is because soybean seed with high raffinose and stachyose concentrations are undesirable and have negative effects on the nutritive value of soymeal and seed consumed by human. Stachyose and raffinose are indigestible by humans and animals, especially monogastric animal such as chicken and pigs, causing flatulence or diarrhea [56]. On the other hand, low raffinose and stachyose levels in soybean seed are desirable [57]. High level of seed sucrose, glucose, and fructose are desirable because it improves taste and flavor of tofu, soymilk, and natto [58]. Currently, soybean cultivars with improved sugar profiles have been released to the market [58], and breeding for desirable sugars in soybean or agricultural practices to improve seed sugars are needed. **3.4. Effect of B and water stress on δ15N (15N/14N ratio) and δ13C (13C/12C ratio)** 

Foliar B did not result in changes in 15N/14N or 13C/12C ratios, but significant differences in these ratios were observed between irrigated and non-irrigated soybean with or without foliar B (Figure 3). The alteration of 15N/14N by increasing 15N (derived from soil nitrogen that is used for nitrate assimilation) and decreasing 14N (derived from atmospheric nitrogen that is used for nitrogen fixation) indicated that the source of nitrogen use changed, and plants favored N from soil over atmospheric nitrogen, indicating that nitrogenase is more sensitive than nitrate reductase under water stress. The mechanisms of this shift are not understood, but Role of Boron Nutrient in Nodules Growth and Nitrogen Fixation in Soybean Genotypes… http://dx.doi.org/10.5772/56994 251

watered plants with B (A) and without B (B); in water stressed plants with B (C) and without B (D); in soybean genotypes in watered plants with B (E) and without B (F); in water stressed plants with B (G) and without B (H); Soybean plants were grown under severe water stress conditions (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. **4. Conclusions Figure 3.** Effects of foliar boron application (1.1 kg B ha-1) on seed δ15N (15N/14N Ratio) (A-C) and δ13C (13C/12C Ratio) (D-H) in soybean genotypes in watered plants with B (A) and without B (B); in water stressed plants with B (C) and without B (D); in soybean genotypes in watered plants with B (E) and without B (F); in water stressed plants with B (G) and without B (H); Soybean plants were grown under severe water stress conditions (soil water potential between – 150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10].

Figure 3. Effects of foliar boron application (1.1 kg B ha-1) on seed δ15N (15N/14N Ratio) (A-C) and δ13C (13C/12C Ratio) (D-H) in soybean genotypes in

Foliar boron application resulted in nodule growth by increasing the number and mass of nodules under well watered or moderate water stress conditions. Also, foliar boron resulted in higher nitrogen fixation and nitrogen assimilation under well watered or moderate water stress conditions. Foliar B application under severe water stress did not enhance nodule number or mass, nitrogen fixation and nitrogen assimilation. This is because severe water stress altered nitrogen and carbon fixation as indicated by changes

sugar fractions by increasing monosaccharides such as glucose and fructose, decreasing sucrose as a result, but increasing both

#### in the values of 15N and 13C natural isotopes. Nitrogen fixation is more sensitive than nitrogen assimilation under severe water stress conditions. Foliar B enhanced seed sugars under well watered conditions, but sever water stress resulted in redistribution of **4. Conclusions**

During carbon fixation by photosynthesis, the naturally occurring stable isotope 13C is discriminated against, and plants would have a smaller 13C to 12C ratio than 13C to 12C ratio in fixed CO2 of the air, suggesting a possible use of this technique to select for water use efficiency (Farquhar et al., 1989*)*. Our results demonstrated that δ 15N and δ13C values changed and enrichment occurred under water stress conditions, suggesting that both nitrogen and carbon metabolism pathways were affected during water stress, impacting seed production and seed

**Figure 2.** Effects of foliar boron application (1.1 kg B ha-1) on nitrogen percentage in leaves and seed in soybean geno‐ types in watered (A,B) and severe water stressed (C,D) soybean genotypes. Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions

**3.4. Effect of B and water stress on δ15N (15N/14N ratio) and δ13C (13C/12C ratio)** 

kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10].

Cultivar Pella Williams 82 Hutcheson Forest

Water stressed plants -B

Cultivar Pella Williams 82 Hutcheson Forest

Nitrogen in leaves Nitrogen in seeds

Watered plants -B

Cultivar Pella Williams 82 Hutcheson Forest

Water stressed plants+B

Cultivar Pella Williams 82 Hutcheson Forest

Nitrogen in leaves Nitrogen in seeds

Watered plants +B

Nitrogen (%)

0

Nitrogen (%)

0

2

4

6

C D

Figure 2. Effects of foliar boron application (1.1 kg B ha-1) on nitrogen percentage in leaves and seed in soybean genotypes in watered (A,B) and severe water stressed (C,D) soybean genotypes. Soybean plants were grown under severe water stress (soil water potential between –150 to –200

Soybean seed sugars are important to soybean seed industry because they determine the quality of seeds beside protein and oil. This is because soybean seed with high raffinose and stachyose concentrations are undesirable and have negative effects on the nutritive value of soymeal and seed consumed by human. Stachyose and raffinose are indigestible by humans and animals, especially monogastric animal such as chicken and pigs, causing flatulence or diarrhea [56]. On the other hand, low raffinose and stachyose levels in soybean seed are desirable [57]. High level of seed sucrose, glucose, and fructose are desirable because it improves taste and flavor of tofu, soymilk, and natto [58]. Currently, soybean cultivars with improved sugar profiles have been released to the market [58], and breeding for desirable sugars in soybean or agricultural practices to improve seed sugars are

Foliar B did not result in changes in 15N/14N or 13C/12C ratios, but significant differences in these ratios were observed between irrigated and non-irrigated soybean with or without foliar B (Figure 3). The alteration of 15N/14N by increasing 15N (derived from soil nitrogen that is used for nitrate assimilation) and decreasing 14N (derived from atmospheric nitrogen that is used for nitrogen fixation) indicated that the source of nitrogen use changed, and plants favored N from soil over atmospheric nitrogen, indicating that nitrogenase is more sensitive than nitrate reductase under water stress. The mechanisms of this shift are not understood, but

8

2

4

6

A B

8

Nitrogen (%)

0

Nitrogen (%)

0

similar to those previously reported [10].

2

4

6

8

2

4

6

8

250 Advances in Biology and Ecology of Nitrogen Fixation

quality.

needed.

raffinose and stachyose due to their possible roles in drought and the acquisition of desiccation tolerance during seed development and maturation. Increasing sugars by foliar B is desirable as high glucose, fructose, and sucrose contribute to soybean seed quality by improving the taste and flavor of soymeal based products such as tofu, soymilk, and natto. Foliar boron application resulted in nodule growth by increasing the number and mass of nodules under well watered or moderate water stress conditions. Also, foliar boron resulted in higher nitrogen fixation and nitrogen assimilation under well watered or moderate water stress conditions. Foliar B application under severe water stress did not enhance nodule number or mass, nitrogen fixation and nitrogen assimilation. This is because severe water stress altered nitrogen and carbon fixation as indicated by changes in the values of 15N and 13C natural isotopes. Nitrogen fixation is more sensitive than nitrogen assimilation under

severe water stress conditions. Foliar B enhanced seed sugars under well watered conditions, but sever water stress resulted in redistribution of sugar fractions by increasing monosacchar‐ ides such as glucose and fructose, decreasing sucrose as a result, but increasing both raffinose and stachyose due to their possible roles in drought and the acquisition of desiccation tolerance during seed development and maturation. Increasing sugars by foliar B is desirable as high glucose, fructose, and sucrose contribute to soybean seed quality by improving the taste and flavor of soymeal based products such as tofu, soymilk, and natto.

Although our research showed that B has beneficial effects on nodules and nitrogen fixation and seed quality, further research is needed to test these findings under field and drought conditions in multi-year and multi-location experiments so that recommendations can be made.

#### **Acknowledgements**

We thank Sandra Mosley for lab assistance and greenhouse management. Also, we thank Leslie Price for his technical assistance on nitrogen and carbon isotopes measurements. The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA's TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportu‐ nity provider and employer.

#### **Author details**

Nacer Bellaloui1 , Alemu Mengistu2 , My Abdelmajid Kassem3 , Craig A. Abel4 and L.H.S. Zobiole5

1 USDA-ARS, Crop Genetics Research Unit, Stoneville, USA

2 USDA-ARS, Crop Genetics Research Unit, Jackson, TN, USA

3 Plant Genomics and Biotechnology Lab, Department of Biological Sciences, Fayetteville State University, Fayetteville, NC, USA

4 Corn Insects and Crop Genetics Research, Iowa State University, Ames, Iowa, USA

5 Crop Protection, R&D Dow AgroSciences – Brazil, Cascavel, Paraná, Brazil

#### **References**

severe water stress conditions. Foliar B enhanced seed sugars under well watered conditions, but sever water stress resulted in redistribution of sugar fractions by increasing monosacchar‐ ides such as glucose and fructose, decreasing sucrose as a result, but increasing both raffinose and stachyose due to their possible roles in drought and the acquisition of desiccation tolerance during seed development and maturation. Increasing sugars by foliar B is desirable as high glucose, fructose, and sucrose contribute to soybean seed quality by improving the taste and

Although our research showed that B has beneficial effects on nodules and nitrogen fixation and seed quality, further research is needed to test these findings under field and drought conditions in multi-year and multi-location experiments so that recommendations can be

We thank Sandra Mosley for lab assistance and greenhouse management. Also, we thank Leslie Price for his technical assistance on nitrogen and carbon isotopes measurements. The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA's TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportu‐

, My Abdelmajid Kassem3

3 Plant Genomics and Biotechnology Lab, Department of Biological Sciences, Fayetteville

4 Corn Insects and Crop Genetics Research, Iowa State University, Ames, Iowa, USA

5 Crop Protection, R&D Dow AgroSciences – Brazil, Cascavel, Paraná, Brazil

, Craig A. Abel4

and

flavor of soymeal based products such as tofu, soymilk, and natto.

made.

**Acknowledgements**

252 Advances in Biology and Ecology of Nitrogen Fixation

nity provider and employer.

, Alemu Mengistu2

State University, Fayetteville, NC, USA

1 USDA-ARS, Crop Genetics Research Unit, Stoneville, USA

2 USDA-ARS, Crop Genetics Research Unit, Jackson, TN, USA

**Author details**

Nacer Bellaloui1

L.H.S. Zobiole5


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## **Symbiotic of Nitrogen Fixation Between Acid Aluminium Tolerant** *Bradyrhizobium japonicum* **and Soybean**

Nisa Rachmania Mubarik and Tedja-Imas Sunatmo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57491

#### **1. Introduction**

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Indonesia is a tropical country in Southeast Asia region, located between the Asia and Australia continents. In most parts of Indonesia, climate variation and high of rainfall causes intensive leaching, soil becomes low content of alkaline and the pH tend to acidic. Indonesia has acid dry land area approximately 102.8 million hectares, but only 55.8 million hectares are suitable for agricultural [1]. The arid lands in Indonesia which are generally formed from mineral soil are acidic (pH 4.6 to 5.5) and poor of nutrients. One effort to increase the soil fertility and plant productivity on acid dry land with planting legumes, such as soybean. Inoculation of root nodule bacteria on soybean plant could enhance soybean quality and its productivity [2 & 3]. Some varieties of acid tolerant soybean, such as Tanggamus, Sibayak, Seulawah, Ratai, and Nanti are issued by the Research Institute for Legumes plants and Tuber Crops Indonesia could grow at acidic soil with pH 4.5-5.0 and produced soybean up to 2000 Kg/hectares on the right growing conditions [4]. Soybeans generally grow in soil at pH 5.5-6.0 while the optimum pH is 6.8. Below pH 4.7 soybean production will decline. It is related to the chemical properties of acid soil, that is high levels of aluminium, high P fixation, iron and manganese concentration increases to the toxic level, sensitive to erosion, and poor biotic status under a low pH conditions [5]. Soybean production could be increase by symbiosis with root nodule bacteria. The effectiveness of symbiotic bacteria in legume root nodules is strongly influenced by the soil conditions. Keyser and Munns [5] suggested that aluminum (Al) with a high concentration (50 µM) is one of the stress factors that can inhibit the growth and prolong the lag phase of root nodulating bacteria. Richardson *et al.* [6] also stated that the Al concentration of 7.5 µM at pH 4.8 can inhibit the expression of nod genes that play a role in nodulation. Furthermore, Johnson and Wood [7] stated that the Al3+ cation can bind to PO4 3- of DNA thereby inhibiting

© 2014 Mubarik and Sunatmo; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DNA replication and transcription. Therefore, strains of acid and high Al-tolerant root nodulating bacteria which have symbiotic effectiveness with soybean are needed to explore.

#### **2. Acid aluminium tolerant** *Bradyrhizobium japonicum*

Some strains of root nodulating bacteria tolerant to acid soil conditions have been reported [8]. The bacteria has ability to fix atmospheric nitrogen (N2) and and convert into ammonium (NH3) [9]. *Bradyrhizobium japonicum* is one of root nodule bacteria that can contribute on soybean growth by providing fixed nitrogen in nodules of soybean plants [2]. *Bradyrhizobi‐ um* is included to the family Rhizobiaceae. This family consists of four genera, namely *Agrobacterium*, *Bradyrhizobium*, *Phyllobacterium*, and *Rhizobium*. The characteristics of *Bradyrhi‐ zobium* are rod-shaped, nonspore-forming cells, motile with one polar or subpolar flagelum, aerobic, Gram-negative, cell-sized of 0.5-0.9 µm and 1.2-3.0 µm, the optimum growth temper‐ ature is 25-30°C at pH 6-7 [10]. *Bradyrhizobium* is known as slow growing bacteria with a generation time ranged 7-20 hours. The bacterial growth on yeast mannitol agar (YMA) needs 5-7 days incubation on room temperature.

*Bradyrhizobium japonicum* has sticky consistency and slimy (mucoid) when grown on media containing carbohydrates. The mucus is an extracellular polysaccharide that serves to maintain bacterial survival in environmental conditions with the concentration of acid and aluminum (Al) is high. Strains of *B. japonicum* have more slimy colony and generally more tolerant on acid-Al stress conditions compared to the dry type colony [8 & 11]. There are not all bacteria categorized as tolerant of acid (pH 4.0-4.5) are also a high Al tolerant. Some strains of *B. japonicum* were tolerant on an acid condition, even at the pH level 4.0-4.5. Twenty five strains of *B. japonicum* has been selected for acid tolerance (pH 4.5) consist of Al 50 µM, Mn 200 µM, Ca 50 µM, and low P 5 µM [12]. One of the *B. japonicum* (BJ) isolate namely BJ 11 wt (wild-type) has the highest tolerance on acid and had a good ability to grow on pH 4.5 media (Figure 1).

**Figure 1.** The growth of *Bradyrhizobium japonicum* BJ11 (wt) on pH 4.5 yeast-extract mannitol agar containing 0.0025% congo red at 10 days incubation in room temperature

Root nodulating bacteria can be distinguished from other bacteria by growing it on media yeast extract mannitol agar (YMA) consist of 10 g/L mannitol, 0.5 g/L K2HPO4, 0.2 g/L MgSO4.7H2O, 0.2 g/L NaCl, and 0.5 g/L yeast extract and containing 0.0025% congo red. Root nodulating bacteria can not absorb congo red or less, and the colony is colorless or pale white [2]. Bradyrhizobia growing on agar media are classified into three types based on the appear‐ ance of colonies, such as: small dry (SD), large mucoid (LM), large watery (LW), and dimor‐ phism [13]. Colony of SD type is round, convex, translucent, and diameter of <1 mm. The LM type is circular, convex, slimy, relatively translucent, and diameter> 1 mm. The LW type is irregular shapes, flat, watery, translucent, and diameter> 1 mm. Dimorphism type strain is called to strain with a mixture of SD and LM type. Colony type can be used to predict tolerant or sensitive strain to acid-Al condition. A small dry colony type strain is more sensitive to acid-Al compare to large one and wet type colony [8]. BJ 11 is the slow-growing colony, circular shape, convex elevation, slimy, translucent, and diameter of colony > 1 mm, it is categorized large mucoid. Other root nodulating bacteria has fast growing and acid producing is classified as genus *Rhizobium*, whereas the slow-growing and alkaline producing reaction belong to the genus *Bradyrhizobium*. The growth reaction on YMA medium which is acidic or alkaline is determined by adding 0.0025% bromothymol blue. Colony of root nodulating bacteria that produce acid reaction is yellow [15].

#### **3. Symbiotic effectiveness**

DNA replication and transcription. Therefore, strains of acid and high Al-tolerant root nodulating bacteria which have symbiotic effectiveness with soybean are needed to explore.

Some strains of root nodulating bacteria tolerant to acid soil conditions have been reported [8]. The bacteria has ability to fix atmospheric nitrogen (N2) and and convert into ammonium (NH3) [9]. *Bradyrhizobium japonicum* is one of root nodule bacteria that can contribute on soybean growth by providing fixed nitrogen in nodules of soybean plants [2]. *Bradyrhizobi‐ um* is included to the family Rhizobiaceae. This family consists of four genera, namely *Agrobacterium*, *Bradyrhizobium*, *Phyllobacterium*, and *Rhizobium*. The characteristics of *Bradyrhi‐ zobium* are rod-shaped, nonspore-forming cells, motile with one polar or subpolar flagelum, aerobic, Gram-negative, cell-sized of 0.5-0.9 µm and 1.2-3.0 µm, the optimum growth temper‐ ature is 25-30°C at pH 6-7 [10]. *Bradyrhizobium* is known as slow growing bacteria with a generation time ranged 7-20 hours. The bacterial growth on yeast mannitol agar (YMA) needs

*Bradyrhizobium japonicum* has sticky consistency and slimy (mucoid) when grown on media containing carbohydrates. The mucus is an extracellular polysaccharide that serves to maintain bacterial survival in environmental conditions with the concentration of acid and aluminum (Al) is high. Strains of *B. japonicum* have more slimy colony and generally more tolerant on acid-Al stress conditions compared to the dry type colony [8 & 11]. There are not all bacteria categorized as tolerant of acid (pH 4.0-4.5) are also a high Al tolerant. Some strains of *B. japonicum* were tolerant on an acid condition, even at the pH level 4.0-4.5. Twenty five strains of *B. japonicum* has been selected for acid tolerance (pH 4.5) consist of Al 50 µM, Mn 200 µM, Ca 50 µM, and low P 5 µM [12]. One of the *B. japonicum* (BJ) isolate namely BJ 11 wt (wild-type) has the highest tolerance on acid and had a good ability to grow on pH 4.5 media (Figure 1).

**Figure 1.** The growth of *Bradyrhizobium japonicum* BJ11 (wt) on pH 4.5 yeast-extract mannitol agar containing

**2. Acid aluminium tolerant** *Bradyrhizobium japonicum*

5-7 days incubation on room temperature.

260 Advances in Biology and Ecology of Nitrogen Fixation

0.0025% congo red at 10 days incubation in room temperature

Effective strains of *Bradyrhizobium japonicum* produce an effective root nodules on their host. Usually one strain of root nodulating bacteria is used as an inoculum for one variety of soybean plant. Selection should be done from large number of tested strains by using a suitable hostplant on soil and climatic conditions of the host habitat [2].

Symbiotic effectiveness is the relative ability of an association between legume and root nodulating bacteria. Effective nodule consist of leghemoglobin, that is an iron-containing red protein binding with O2 that controls the partial pressure of O2 (pO2) in the nodule [15]. When pO2 was below or above normal condition (0.21 atm), it could decrease the activity of N2 fixation. Leghemoglobin is induced by the interaction between *Bradyrhizobium* with soybean.

Effective nodule tends to be large size, reddish, and able to fix nitrogen gas from air. In addition, the effective root nodules have a limited number and distribution, usually found on the main root and secondary first root [14]. Ineffective nodules tend to be small, numerous, greenish white (pale), unable to fix nitrogen from air and spread the root system [14].

Symbiotic effectiveness of acid-tolerant soybean with acid-Al tolerant *B. japonicum* could be done by using Leonard bottle modified that consists of two volumes of 700 ml bottles of ketchup. One bottle is cut at the base and used for growth media that contains sand and charcoal. Other bottle cut at the neck and is used as a reservoir for the nutrient solution [16]. The lower bottle is filled with 300 ml of N-free nutrient solution of pH 4.5 [17] and 100 ml of N-free nutrient solution poured into growing medium in the mixture form of sand and coconut shell charcoal about 480 gram. Before used, sand is sieved and washed

with clean water several times until clean and dry. Each bottle is covered with cement paper and sterilized by autoclaving at 121°C and 1 atmosphere for 2 hours. Two days sprouts of soybean in Leonard jar. Each sprout was inoculated with 108 cells ml-1 of *B. japonicum*. N-free nutrient solution and nutrient solution contained 5 mM KNO3 used as control treatments, respectively. Symbiotic effectiveness value (SE) is measured based on percentage of dry weight of plants inoculated with tested strain toward dry weight of plants treated with KNO3 or reference strain. *Bradyrhizobium japonicum* USDA 110 is used as a reference strain and completely genomic sequenced [18].

The effectiveness of symbiosis can be observed in several ways viz. the determination of plant dry weight, total N content, and nitrogenase activity [2]. Dry weight of the plant is still considered relevant for evaluating the effectiveness of symbiotic root nodulating bacteria with soybean plants, because plant dry weight significantly correlated with total N content [14]. Plant dry weight is usually correlated with the dry weight of root nodules. Upper plant dry weight is used as a parameter to evaluate the binding of N, because as much as 70% of the fixing N is transported to the upper plant [14].

The symbiotic interaction between soybean and root nodulating bacteria played an important role in increasing the plant growth of soybean plant. Effectiveness of a root nodulating bacteria in fixing nitrogen is affected by the compatibility between bacteria and the soybean plant. Mubarik *et al*. [19] described that inoculation of BJ 11 (wt) root nodulating bacteria could increase the height of soybean plant and shoot dry weight until 37 days after planting (DAP) (Figure 2). The nodule dry weight was positively correlated with the ability of plants to fix N and shoot dry weight. The value of symbiotic effectiveness, shoot dry weight, and N uptake of BJ 11 was higher than USDA 110 as reference strain (Table 1).

**Figure 2.** Soybean plant (37 days after planting) grow on a Leonard bottle using N-free nutrient solution pH 4.5 + Al 50 µM: (1) without inoculation BJ 11 and (2) inoculated with BJ 11 [16]


0 =no detection, N:without BJ inoculation consist of 5 mM KNO3, N0: without BJ inoculation and without 5mM KNO3, Symbiotic Effectiveness (SE) against N/R.

**Table 1.** Effect of inoculation of *B. japonicum* on soybean cultivar Slamet at 37 DAP using N-free solution at pH 4,5 + Al 50 µM (Mubarik *et al.* 2012)

*Bradyrhizobium japonicum* is able to form nodules and fix nitrogen. Nodule formation on the roots of leguminous plants generally through the following stages: (i) the introduction of a suitable partner on the part of the plant and bacterial attachment to root hairs, (ii) the hair root invasion by bacteria through thread-forming infection threads, (iii) the bacteria moves to the main root through infection threads, (iv) the formation of bacteria in plant cells called bacte‐ roids, and (v) plant and bacterial cell division that is constantly and will produce the mature root nodules [15]. Stages of nodulation (nodule formation) is controlled by the *nod* genes.

The source of energy for nitrogen fixation in bacteroids depends on host photosynthate which is transported through the membrane simbiosome in the form intermediate product of the tricarboxylic acid cycle (Krebs cycle) such as succinic acid, fumaric and malic acid which is a electron donor to produce ATP and reduce N2. Pyruvic acid is the reductant that involved directly as an electron donor in the nitrogenase system [15]. The N2 binding reaction that occurs in bacteroids as follows:

$$\text{N}\_2 + 8\text{e} + 8\text{H}^\* + 16\text{MgATP} \overset{\text{nitrogen}}{\rightarrow} 2\text{NH}\_3 + \text{H}\_2 + 16\text{MgADP} + 16\text{ Pi}$$

Complex of nitrogenase reduces the triple bond of N2 into ammonia molecules. Nitrogenase enzyme activity can be measured by the acetylene reduction technique. Acetylene (C2H2) can be used as an alternative substrat to N2. Reduction of N2 and acetylene by nitrogenase as follows:

N2+ 12 ATP + 6e- + 6H+→2 NH3+ 12 ADP + 12 Pi

C2H2+ 4 ATP + 2e- + 2H+→C2H4+ 4ADP + 4Pi

with clean water several times until clean and dry. Each bottle is covered with cement paper and sterilized by autoclaving at 121°C and 1 atmosphere for 2 hours. Two days sprouts of soybean in Leonard jar. Each sprout was inoculated with 108 cells ml-1 of *B. japonicum*. N-free nutrient solution and nutrient solution contained 5 mM KNO3 used as control treatments, respectively. Symbiotic effectiveness value (SE) is measured based on percentage of dry weight of plants inoculated with tested strain toward dry weight of plants treated with KNO3 or reference strain. *Bradyrhizobium japonicum* USDA 110 is used as a

The effectiveness of symbiosis can be observed in several ways viz. the determination of plant dry weight, total N content, and nitrogenase activity [2]. Dry weight of the plant is still considered relevant for evaluating the effectiveness of symbiotic root nodulating bacteria with soybean plants, because plant dry weight significantly correlated with total N content [14]. Plant dry weight is usually correlated with the dry weight of root nodules. Upper plant dry weight is used as a parameter to evaluate the binding of N, because as much as 70% of the

The symbiotic interaction between soybean and root nodulating bacteria played an important role in increasing the plant growth of soybean plant. Effectiveness of a root nodulating bacteria in fixing nitrogen is affected by the compatibility between bacteria and the soybean plant. Mubarik *et al*. [19] described that inoculation of BJ 11 (wt) root nodulating bacteria could increase the height of soybean plant and shoot dry weight until 37 days after planting (DAP) (Figure 2). The nodule dry weight was positively correlated with the ability of plants to fix N and shoot dry weight. The value of symbiotic effectiveness, shoot dry weight, and N uptake

**Figure 2.** Soybean plant (37 days after planting) grow on a Leonard bottle using N-free nutrient solution pH 4.5 + Al

reference strain and completely genomic sequenced [18].

of BJ 11 was higher than USDA 110 as reference strain (Table 1).

50 µM: (1) without inoculation BJ 11 and (2) inoculated with BJ 11 [16]

fixing N is transported to the upper plant [14].

262 Advances in Biology and Ecology of Nitrogen Fixation

The comparison between the substrate N2 reduction by C2H2 is 3:1, and according to calculation [20] the total amount of N fixed by plants (µg) = µmol C2H4 x 28.

While the C2H2 reduction can provide a useful tool for detecting N2-fixing activity in both legumes and non-legumes plants, the method is unsuitable for measuring N2 fixation at field scales. There are some suitability of methods for quantifying N2 fixation for crop legumes, such as measurement of N difference, relative ureide method, 15N natural abundance, and 15N isotope dilution [21]. But none of the methods for assessing N2 fixation is perfect. Some additional informations are needed to support the N2 fixation data, such as assessment of nodulation, growth analysis, rooting patterns of N2 fixing and companion non N2-fixing plants, determination of mineral N soil, and soil analysis [21].

### **4. Greenhouse experiments of symbiotic between acid aluminium tolerant** *B. japonicum* **and soybean on acid soils**

Situmorang *et al*. [22] prepared media for soybean cultivication by using mixed composition of 1200 g acid soil (pH 4.5) and 800 g peat in a polybag. Peat is used as an additional organic matter to the soil. Acid soils and peat are prepared by drying and filtering using 2 mm pore of diameter. The media is sterilized by autoclave at 121˚C and 2 atm for one hour. The media is inoculated with 20% (v/w) of 108 cells/ml bacterial culture. Positive control media is added with 5 mM KNO3. Plant harvest are divided into two groups, at the 50 DAP to crop nodules and 75-108 DAP to crop pods of legume. Three isolates are used viz. BJ 11 (wt), and its mutant BJ 11(5) and BJ 11(19). Wahyudi *et al.* [23] has been constructed several strains of acidaluminium tolerant *B. japonicum* with increased symbiotic effectiveness through transposon TN5 mutagenesis, such as BJ 11(5), BJ 11(19), BJ 11 (20), and KDR 15 (37). The mutants could grow better on acid pH (4.0-4.5) and when each mutant inoculated to soybean plants will influenced better of symbiotic effectiveness, plant height, shoot and root weight, number of flowers, pods, dry weight of 100 seeds, and plant N-content [22].

Inoculation of BJ 11(19) isolate increased number of seeds and pods higher than the other treatments [22]. Acid tolerant soybean such as Slamet generally has weight 12.5 g of 100 seeds [24]. BJ 11 (19) showed the highest 13.5 g of 100 seeds. Pods that were already formed then were filled with photosynthate to form seeds. Numbers of seeds are effected by the number and size of pods. Higher number of pods also produce higher numbers of seeds [25].

Further experiments are done in acid soil plots (pH 4.5). Totally 12 plot experiments, each plot measured 1 m x 2 m x 0.2 m filled with 45 kg of acid soils (pH 4.5) and 10% (w / w) peat or rice husk as innoculant carrier. Each plot planting with soybean sprouts each with a spacing of 20 x 40 cm2 . Amount of inoculant (about 1.0 x 108 cells/ml) in peat-carrier is applied to each plot. Every hole on plot planted with 5 seedling soybeans and to be reduced to 3 plants at 30 days after planting. Each plot is separated by a distance of 1 m from other plot. Results of plot experiment showed that the effectiveness of symbiotic BJ 11 (19) with soybean is significantly had higher value on the plant height, dry weight of upper crop, root nodules, nodule number, nitrogenase activity, and weight of 100 seeds. Treatment of compost before planted soybean in acid soils could produce better crops and increase producing of soybean seeds compare to without compost (Figure 3). The compost consists of plant residues and soil microbes that can improve acid soil structure becomes more fertile and porous.

While the C2H2 reduction can provide a useful tool for detecting N2-fixing activity in both legumes and non-legumes plants, the method is unsuitable for measuring N2 fixation at field scales. There are some suitability of methods for quantifying N2 fixation for crop legumes, such as measurement of N difference, relative ureide method, 15N natural abundance, and 15N isotope dilution [21]. But none of the methods for assessing N2 fixation is perfect. Some additional informations are needed to support the N2 fixation data, such as assessment of nodulation, growth analysis, rooting patterns of N2 fixing and companion non N2-fixing plants,

**4. Greenhouse experiments of symbiotic between acid aluminium tolerant**

Situmorang *et al*. [22] prepared media for soybean cultivication by using mixed composition of 1200 g acid soil (pH 4.5) and 800 g peat in a polybag. Peat is used as an additional organic matter to the soil. Acid soils and peat are prepared by drying and filtering using 2 mm pore of diameter. The media is sterilized by autoclave at 121˚C and 2 atm for one hour. The media is inoculated with 20% (v/w) of 108 cells/ml bacterial culture. Positive control media is added with 5 mM KNO3. Plant harvest are divided into two groups, at the 50 DAP to crop nodules and 75-108 DAP to crop pods of legume. Three isolates are used viz. BJ 11 (wt), and its mutant BJ 11(5) and BJ 11(19). Wahyudi *et al.* [23] has been constructed several strains of acidaluminium tolerant *B. japonicum* with increased symbiotic effectiveness through transposon TN5 mutagenesis, such as BJ 11(5), BJ 11(19), BJ 11 (20), and KDR 15 (37). The mutants could grow better on acid pH (4.0-4.5) and when each mutant inoculated to soybean plants will influenced better of symbiotic effectiveness, plant height, shoot and root weight, number of

Inoculation of BJ 11(19) isolate increased number of seeds and pods higher than the other treatments [22]. Acid tolerant soybean such as Slamet generally has weight 12.5 g of 100 seeds [24]. BJ 11 (19) showed the highest 13.5 g of 100 seeds. Pods that were already formed then were filled with photosynthate to form seeds. Numbers of seeds are effected by the number

Further experiments are done in acid soil plots (pH 4.5). Totally 12 plot experiments, each plot measured 1 m x 2 m x 0.2 m filled with 45 kg of acid soils (pH 4.5) and 10% (w / w) peat or rice husk as innoculant carrier. Each plot planting with soybean sprouts each with a spacing of 20

Every hole on plot planted with 5 seedling soybeans and to be reduced to 3 plants at 30 days after planting. Each plot is separated by a distance of 1 m from other plot. Results of plot experiment showed that the effectiveness of symbiotic BJ 11 (19) with soybean is significantly had higher value on the plant height, dry weight of upper crop, root nodules, nodule number, nitrogenase activity, and weight of 100 seeds. Treatment of compost before planted soybean in acid soils could produce better crops and increase producing of soybean seeds compare to

. Amount of inoculant (about 1.0 x 108 cells/ml) in peat-carrier is applied to each plot.

and size of pods. Higher number of pods also produce higher numbers of seeds [25].

determination of mineral N soil, and soil analysis [21].

264 Advances in Biology and Ecology of Nitrogen Fixation

*B. japonicum* **and soybean on acid soils**

flowers, pods, dry weight of 100 seeds, and plant N-content [22].

x 40 cm2

### **5. Viability test of acid-aluminium** *B. japonicum* **inoculant using peat as carrier**

Viability of *B. japonicum* shoud be tested before used as an inoculant on fields experiments. Handayani *et al.* [26] conducted to test the viability of strains of acid-aluminium tolerant after a period of storages (1, 2, and 3 months) both at room temperature (± 25 °C) and 10 °C. The inoculant of *B. japonicum* BJ 11 (wt), and its mutants viz. BJ 11 (5) and BJ 11 (19), were tested by using sterilized peat as carrier (Figure 4). Peat is an decaying-organic material containing humic acid and organic-C and N which suitable for microbial growth. The result of viability test showed that there were an interaction between strain types, temperature, and a period of storage. The Inoculant of BJ 11 (19) which was stored at temperature 10 °C for 2 months showed the highest viability at 2,5 x 108 cell/g inoculants (Table 2).

**Figure 3.** Growth of acid tolerant soybean variety Slamet 38 DAP on plot experiments: (1) control without inoculation, (2) control without inoculation + compost, (3) inoculation with BJ 11 (wt), (4) inoculation with BJ 11 (wt) + compost, (5) inoculation with BJ 11 (19), (6) inoculation with BJ 11 (wt) + compost.


Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05)

**Table 2.** Viability of three acid aluminium tolerant *B. japonicum* strains (cell. g-1) stored at room temperature (± 25 °C) and 10 °C at 3 months storage [26]

**Figure 4.** The formula of inoculant acid-aluminium tolerant *B. japonicum* containing 109 cells g-1 using peat as carrier. Each pack contains 0.5 kg of inoculant for 10 kg of soybean seeds

#### **6. Field trial of application of acid-aluminium tolerant** *B. japonicum* **on soybean**

There are three locations for field trials to apply of the formula acid-aluminium tolerant *B. japonicum* on soybean viz. Jasinga (West Java), Sukadana (Province Lampung), and Tambang Ulang (Province South Kalimantan). Planting sites prepared a total area 1 hectare. Before planting on the field, the chemical contents of the soil and total plate count of soil bacteria are analyzed (Table 3 & 4). There are not found indigenous *B. japonicum* on all of field trial locations before symbiotic effectiveness treatments.


**Table 3.** Chemical properties of soil at the field trial locations

**Strain Temperature**

266 Advances in Biology and Ecology of Nitrogen Fixation

BJ 11 (5)

BJ 11 (19)

BJ11 (wt)

**soybean**

Range Test (α = 0.05)

and 10 °C at 3 months storage [26]

**Storage periode (months) 1 2 3**

Room 9.8 x 107 cdef 2.8 x 107 f 1.3 x 108 abcdef 10 ºC 1.4 x 108 abcdef 1.2 x 108 abcdef 7.6 x 107 def

Room 2.4 x 108 ab 2.0 x 108 abc 1.1 x 108 bcdef 10 ºC 1.8 x 108 abcd 2.5 x 108 a 1.8 x 108 abcd

Room 1.6 x 108 abcde 1.1 x 108 bcdef 4.2 x 107 ef 10 ºC 1.3 x 108 abcdef 1.1 x 108 bcdef 1.9 x 108 abcd

Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple

**Table 2.** Viability of three acid aluminium tolerant *B. japonicum* strains (cell. g-1) stored at room temperature (± 25 °C)

**Figure 4.** The formula of inoculant acid-aluminium tolerant *B. japonicum* containing 109 cells g-1 using peat as carrier.

**6. Field trial of application of acid-aluminium tolerant** *B. japonicum* **on**

There are three locations for field trials to apply of the formula acid-aluminium tolerant *B. japonicum* on soybean viz. Jasinga (West Java), Sukadana (Province Lampung), and Tambang

Each pack contains 0.5 kg of inoculant for 10 kg of soybean seeds


**Table 4.** Total plate count of bacteria and total of indigenous *B. japonicum* isolated from soil on planting sites

The field trial was conducted to examine the efficiency of BJ 11 (wt) and BJ 11 (19) on the growth, nodulation and yield of soybean variety Tanggamus and Anjasmoro. Tanggamus is one of leading variety which can adapt to dry acid soil, Anjasmoro generally showed good adaptation on paddy fields.

The seeds were coated with the inoculum formula before sowing. Seeds were sown by hand in each hole and planted 3 seeds per hole at a depth of 3 cm, distance of hole 20 cm x 40 cm. Fertilizer was placed at other hole besides of seeds hole. Watering was carried regularly if no rain. Removal of weeds or grasses are done as far as possible.

Soybean seed are sown by hand in a hole at soil. There were three seeds per polybag. Soybean seeds were selected based on the same size and healthy (able to shoot). Some treatments were conducted to soybean seed as follows: 1. inoculated by *B. japonicum* galur BJ 11, 2. inoculated by BJ 11 and application with 100 % N fertilizer; 3. inoculated by *B. japonicum* galur BJ 11 and application with 50 % N fertilizer + 50% compost; 4. Control treatment: without inoculant, without inoculant + 100% N fertilizer, without inoculant + 50 % N fertilizer + 50% compost.

Each treatments were done at 150-200 m2 and replicated two times per treatment. Mineral fertilization 100% N treatment consisted of 100 Kg ha-1 urea + 200 Kg ha-1 TSP (trisodium phosphate) + 100 Kg ha-1 KCl. For 50% N consisted of a half dose of urea + 200 Kg ha-1 TSP + 100 Kg ha-1 KCl + compost 1000 Kg ha-1. Compost was spread out at land surface one week before seeds planting. The compost only consisted of decaying plants and decomposed by microbes. There are not found rhizobia in compost, and consist of phosphate solubilizing bacteria as much as 320 cell.ml-1. Urea used twice at one planting period viz a half dose at seeds planting and the rest at 30 days after planting (DAP) [27].

Growth parameters such as plant height at 30 days after planting (DAP), number of pods at 90 DAP, total number of seeds, total of seed weight, and weight of 100 seeds numbers of pods compare to control were determined. Growth parameters were measured from 10 plants per treatments. Data were analyzed using completely randomized design and the means at p<0.05 level of significance.

The results of field experiments showed that there were a significant effect of *B. japonicum* inoculation for soybean variety Tanggamus and Anjasmoro which grown at Jasinga –West Java, Tanah Laut-South Kalimantan and Sukadana-Lampung compared to control, without inoculants and fertilizer (Table 5, 6 & 7). Inoculation BJ 11 formula showed a better response on soybean growth than control, treatment without fertilizer and inoculant. Plants inoculated with BJ 11 (wt) and its mutant BJ 11 (19) showed higher plant height, number of pods, and seeds, weight of 100 seeds compare to control. To improve field-scale of soybean production in acid soils still need N- fertilizer, but the application of inoculant *B. japonicum* can reduce a half of N fertilizer.



application with 50 % N fertilizer + 50% compost; 4. Control treatment: without inoculant, without inoculant + 100% N fertilizer, without inoculant + 50 % N fertilizer + 50% compost.

fertilization 100% N treatment consisted of 100 Kg ha-1 urea + 200 Kg ha-1 TSP (trisodium phosphate) + 100 Kg ha-1 KCl. For 50% N consisted of a half dose of urea + 200 Kg ha-1 TSP + 100 Kg ha-1 KCl + compost 1000 Kg ha-1. Compost was spread out at land surface one week before seeds planting. The compost only consisted of decaying plants and decomposed by microbes. There are not found rhizobia in compost, and consist of phosphate solubilizing bacteria as much as 320 cell.ml-1. Urea used twice at one planting period viz a half dose at seeds

Growth parameters such as plant height at 30 days after planting (DAP), number of pods at 90 DAP, total number of seeds, total of seed weight, and weight of 100 seeds numbers of pods compare to control were determined. Growth parameters were measured from 10 plants per treatments. Data were analyzed using completely randomized design and the means at p<0.05

The results of field experiments showed that there were a significant effect of *B. japonicum* inoculation for soybean variety Tanggamus and Anjasmoro which grown at Jasinga –West Java, Tanah Laut-South Kalimantan and Sukadana-Lampung compared to control, without inoculants and fertilizer (Table 5, 6 & 7). Inoculation BJ 11 formula showed a better response on soybean growth than control, treatment without fertilizer and inoculant. Plants inoculated with BJ 11 (wt) and its mutant BJ 11 (19) showed higher plant height, number of pods, and seeds, weight of 100 seeds compare to control. To improve field-scale of soybean production in acid soils still need N- fertilizer, but the application of inoculant *B. japonicum* can reduce a

> **Plant height at 90 DAP (cm)**

BJ 11 (19) + 1 N 30.6 d 1 c 28.2 cd 34.2 c 1.5 c 10.8 c 17.5 d 2.83 cd 13.42 b BJ 11 (19) + 1/2 N + C 35.8 b 0.7 cd 31.2 bc 41 b 2.6 a 16.2 b 29.4 bc 4.33 bc 12.97 b BJ 11 (19) 31.9 cd 0.3 de 19.9 e 37.5 bc 1.4 c 10 c 18.3 d 2.27 de 12.60 b BJ 11 (WT) + 1 N 40.1 a 1.7 b 35.4 b 46.5 a 2.3 ab 19.7 b 35 b 4.55 b 13.21 b BJ 11 (WT) + 1/2 N + C 42 a 2.3 a 45.2 a 51.9 a 2.5 a 31.3 a 51.1 a 6.29 a 12.55 b BJ 11 (WT) 33.6 bc 0.4 de 21.8 de 34.c1 0.1 d 5.5 c 7.1 e 1.02 e 15.06 a 1 N 41.3 a 1.6 b 36.4 b 38.1 bc 1.6 bc 16.6 b 25,5 bcd 3.43 bcd 12.75 b 1/2 N + C 42.2 a 2.1 ab 29.9 bc 38.1 bc 1.7 bc 17.1 b 29.8 bc 4.23 bc 13.17 b Control 26.4 e O e 20.5 e 34.7 c 1.8 bc 10.9 c 21.8 cd 2.95 cd 13.26 b

**Number of branch at 90 DAP**

**Number of pods**

**Number of seed**

**Total of seed weight (g)**

**Weight of 100 seeds (g)**

and replicated two times per treatment. Mineral

Each treatments were done at 150-200 m2

268 Advances in Biology and Ecology of Nitrogen Fixation

level of significance.

half of N fertilizer.

**Plant height at 45 DAP (cm)**

**Number of branch** **Number of flower**

**Anjasmoro**

**Treatment**

planting and the rest at 30 days after planting (DAP) [27].

BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05).


BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05).

**Table 5.** Growth of Anjasmoro and Tanggamus cultivar soybean plants on treatment with acid-aluminium tolerant *B. japonicum* formula on acid soil at Jasinga- West Java



BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N =

50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer

(NPK) and inoculants. Numbers on the same column followed by the same letter were not

significantly different based on Duncan Multiple Range Test (α = 0.05).


BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N =

50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer

(NPK) and inoculants. Numbers on the same column followed by the same letter were not

significantly different based on Duncan Multiple Range Test (α = 0.05).

**Table 6.** Growth of Anjasmoro and Tanggamus cultivar soybean plants on treatment with acid-aluminium tolerant *B. japonicum* formula on acid soil at Tambang Ulang-South Kalimantan


**Anjasmoro**

**Plant height at 30 DAP (cm)**

270 Advances in Biology and Ecology of Nitrogen Fixation

**Plant height at 90 DAP (cm)**

significantly different based on Duncan Multiple Range Test (α = 0.05).

Number of leaf at 90 DAP

Plant height at 90 DAP (cm)

> 59.4 ab

significantly different based on Duncan Multiple Range Test (α = 0.05).

*japonicum* formula on acid soil at Tambang Ulang-South Kalimantan

Plant height at 30 DAP (cm)

30.4 cd

33.8 ab

33.9 ab

**Numbe r of leaf at 90 DAP**

**Number of branch at 30 DAP**

BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not

> Number of branch at 30 DAP

Number of branch at 90 DAP

BJ 11 (19) + 1 N 28.6 d 62.5 a 32.9 a 1.2 ab 3 ab 37.9 abc 49.3 bcd 4.3 bc 11.0 ab

BJ 11 (19) 51.1 c 50.5 c 18.5 c 1.5 a 2.2 c 21.8 e 47.1 cd 4.8 bc 10.3 b

1 N 34.9 a 45.8 c 23.3 bc 1.9 a 2.5 bc 21.7 e 36.7 d 3.5 c 10.3 b 1/2 N + C 32.4 bc 45.7 c 21.1 bc 1.7 a 2.7 bc 19.8 e 38.8 d 4.1 c 11.4 ab Control 28.8 d 48.9 c 22.7 bc 0.5 c 3.5 a 26.3 de 50.3 bcd 4.4 bc 10.6 b

**Table 6.** Growth of Anjasmoro and Tanggamus cultivar soybean plants on treatment with acid-aluminium tolerant *B.*

BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not

Number of pod at 90 DAP

24.8 b 1.5 a 2.7 bc 33.6 bcd 56.2 bc 4.9 bc 10.4 b

57.3 b 31.8 a 1.7 a 3.2 ab 41.1 ab 74.9 a 6.9 a 11.4 ab

55.4 b 25 b 0.6 bc 2.6 bc 31.6 cd 64.4 ab 5.8 ab 12.1 a

35 a 59 ab 32.6 a 1.7 a 2.7 bc 45.1 a 64.9 ab 6.8 a 10.9 ab

Number of seed

Total of seed weight (g)

**Number of branch at 90 DAP**

BJ 11 (WT) 40.9 cd 69.9 a 32 ab 3.3 a 3.6 a 67.7 a 101.1 ab 17.5 ab 13.3 c 1 N 53.4 a 50.8 cd 21.6 ef 3 ab 2.9 bc 27.9 cd 46.3 d 6 e 16.1 a 1/2 N + C 35.8 e 47.6 d 21.2 ef 2.3 bc 3.1 ab 27.2 cd 63.1 cd 9.2 de 13.3 c Control 41 cd 45.8 d 17.3 f 3.2 a 2.4 cd 15.6 d 40.6 d 5 e 14.6 abc

**Number of pod at 90 DAP**

44. 3 b 71.2 a 29 abc 2 c 3 ab 58.2 a 75.9 c 12.3 cd 15.3 ab

**Number of seed**

**Total of seed weight (g)**

**Weight of 100 seeds (g)**

Weight of 100 seeds (g)

**Treatment**

N + C

BJ 11 (WT) + 1/2

Tanggamus

Treatment

+ C

N + C

BJ 11 (WT)

BJ 11 (19) + 1/2 N

BJ 11 (WT) + 1 N

BJ 11 (WT) + 1/2

BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05).


BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05).

**Table 7.** Growth of Anjasmoro and Tanggamus variety soybean plants on treatment with acid-aluminium tolerant *B. japonicum* formula on acid soil at Sukadana- Lampung

#### **7. Conclusion**

Effectiveness symbiotic between soybean and acid-toleran aluminium root nodule bacteria, such as *Bradyrhizobium japonicum* BJ 11 played an important role on increasing the plant growth on acid soil (pH> 4.5). The bacteria provided fixed nitrogen to soybean plant and then support growth and development of plants. Soybean plants inoculated with *B. japonicum* strain BJ 11 (wild-type) and its mutant BJ 11 (19) showed better growth than control without inoculation in greenhouse and field trial experiments. B. japonicum inoculant on peat as carrier showed high viability and stability during storages.

#### **Acknowledgements**

This project was supported by Integrated Excelence Research, The Ministry of Research and Technology, Republic of Indonesia in 1996 to TI Sunatmo and Incentive Programs for Applied Research, The Ministry of Research and Technology, Republic of Indonesia in 2007-2009 to NR Mubarik. We thank our colleaguaes and students for support in the project.

#### **Author details**

Nisa Rachmania Mubarik and Tedja-Imas Sunatmo

\*Address all correspondence to: nrachmania@ipb.ac.id

Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Agatis, IPB Dermaga, Bogor, Indonesia

#### **References**


[4] Suhartina. Perkembangan dan Deskripsi Varietas Unggul Kedelai 1998-2002. (*Devel‐ opment and Description of Soybean Varieties 1998-2002*). Malang: Crops Research Insti‐ tute for Legumes and Tubers; 2003.

**7. Conclusion**

high viability and stability during storages.

272 Advances in Biology and Ecology of Nitrogen Fixation

Nisa Rachmania Mubarik and Tedja-Imas Sunatmo

\*Address all correspondence to: nrachmania@ipb.ac.id

University, Jalan Agatis, IPB Dermaga, Bogor, Indonesia

University of Hawaii; 1994.

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*acid dryland*) Sinar Tani 2006; Edition 24-30 Mei 2006.

**Acknowledgements**

**Author details**

**References**

Effectiveness symbiotic between soybean and acid-toleran aluminium root nodule bacteria, such as *Bradyrhizobium japonicum* BJ 11 played an important role on increasing the plant growth on acid soil (pH> 4.5). The bacteria provided fixed nitrogen to soybean plant and then support growth and development of plants. Soybean plants inoculated with *B. japonicum* strain BJ 11 (wild-type) and its mutant BJ 11 (19) showed better growth than control without inoculation in greenhouse and field trial experiments. B. japonicum inoculant on peat as carrier showed

This project was supported by Integrated Excelence Research, The Ministry of Research and Technology, Republic of Indonesia in 1996 to TI Sunatmo and Incentive Programs for Applied Research, The Ministry of Research and Technology, Republic of Indonesia in 2007-2009 to NR

Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural

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[20] Masterson CL, Murphy PM. The acetylene reduction technology. In: Subba Rao NS, editor. Recent Advance in Biological Nitrogen Fixation. New Delhi: Oxford and IBH

[21] Unkovich M, Herridge D, Peoples M, Cadisch G, Boddey B, Giller K, Alves B, Chalk P. Measuring Plant-associated Nitrogen Fixation in Agricultural Systems. Canberra:

[22] Situmorang ARF, Mubarik NR, Triadiati. The use of acid-alumunium tolerant *Bradyr‐ hizobium japonicum* inoculant for soybean grown on acid soils. Hayati J Biosci

[23] Wahyudi AT, Suwanto A, Tedja-Imas, Tjahyoleksono A. Screening of acid- alumuni‐ um tolerant *Bradyrhizobium japonicum* strain analysis of marker genes and competi‐

[24] Sunarto. 1995. Pemuliaan kedelai untuk toleran terhadap tanah masam dan keracun‐ an Al. (*The breeding soybeans for tolerance to acid soils and Al toxicity*). J Indust Pangan

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[27] Mubarik NR, Imas T, Wahyudi AT, Triadiati, Suharyanto, Widiastuti H. The use of acid-alumunium tolerant *Bradyrhizobium japonicum* formula for soybean grown on field acid soil.On line: World Academy of Science, Engineering and Technology 2011; 53: 879-882 (eISSN 2010-3778). Printed: World Academy of Science, Engineering and

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### *Edited by Takuji Ohyama*

Biological nitrogen fixation has essential role in N cycle in global ecosystem. Several types of nitrogen fixing bacteria are recognized: the free-living bacteria in soil or water; symbiotic bacteria making root nodules in legumes or non-legumes; associative nitrogen fixing bacteria that resides outside the plant roots and provides fixed nitrogen to the plants; endophytic nitrogen fixing bacteria living in the roots, stems and leaves of plants. In this book there are 11 chapters related to biological nitrogen fixation, regulation of legume-rhizobium symbiosis, and agriculture and ecology of biological nitrogen fixation, including new models for autoregulation of nodulation in legumes, endophytic nitrogen fixation in sugarcane or forest trees, etc. Hopefully, this book will contribute to biological, ecological, and agricultural sciences.

Photo by Hans Verburg / iStock

ISBN 978-953-51-1216-7 ISBN 978-953-51-4238-6 Advances in Biology and Ecology of Nitrogen Fixation

Advances in Biology and

Ecology of Nitrogen Fixation

*Edited by Takuji Ohyama*